Polymeric systems and uses thereof in theranostic applications

ABSTRACT

Polymeric systems useful for theranostic applications are disclosed. The polymeric systems comprise a fluorescent or fluorogenic moiety and a therapeutically active agent, each attached to the same or different polymeric moiety. The polymeric systems are designed such that a fluorescent signal is generated in response to a chemical event, preferably upon contacting an analyte (e.g., an enzyme) that is over-expressed in a diseased tissue or organ. Probes useful for inclusion in such polymeric systems, processes of preparing such probes and the polymeric systems, and uses thereof in diagnostic and/or theranostic applications are also disclosed.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.15/124,360 filed on Sep. 8, 2016, which is a National Phase of PCTPatent Application No. PCT/IL2015/050269 having International FilingDate of Mar. 13, 2015, which claims the benefit of priority under 35 USC§ 119(e) of U.S. Provisional Patent Application No. 61/952,259 filed onMar. 13, 2014. The contents of the above applications are allincorporated by reference as if fully set forth herein in theirentirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to therapyand diagnosis (theranostic) and, more particularly, but not exclusively,to polymeric systems in which a labeling moiety (e.g., a fluorescent orfluorogenic moiety) or a labeling moiety and a therapeutically activeagents are attached to a polymeric backbone, and to uses thereof indiagnostic and theranostic applications.

In the past few years, tremendous efforts have been employed inmonitoring cancer treatment, detecting response to drugs and measuringreal-time accumulation of the drug within the tumor. Numerousnanocarrier systems have been developed (e.g., polymers, liposomes,micelles, dendrimers, etc.) and studied as delivery vehicles foranticancer drugs to improve the drugs' biodistribution, solubility, andhalf-life, and thus to exhibit enhanced efficacy and reduced toxicity.Clinically-available fluorescence-based imaging contrast agents (e.g.,indocyanine green and fluorescein) hold many of the limitationsattributed to chemotherapeutic agents, including low molecular weight,short half-life and poor selectivity. Consequently, monitoring slowprocesses, such as drug accumulation at the tumor site, is challenging.

Combining therapeutic and diagnostic modalities on the same deliverysystem, thereby forming a theranostic (therapy and diagnostic)nanomedicine, may overcome these limitations, while enablingsimultaneous monitor and treatment of angiogenesis-dependent diseases,like cancer [Kelkar, S. S. and T. M. Reineke, Theranostics: CombiningImaging and Therapy. Bioconjug Chem, 2011. 22(10): p. 1879-1903].Information obtained from theranostic nanomedicine is exploited for finetuning the therapeutic dose, while monitoring the progression of thediseased tissue, treatment efficacy and delivery kinetics [Janib et al.Adv Drug Deliv Rev, 2010. 62(11): p. 1052-1063; McCarthy, J. R., Thefuture of theranostic nanoagents. Nanomedicine, 2009. 4(7): p. 693-695].This, from a clinical prospective, should enhance early diagnosis andtreatment and may decrease drugs under- or over-dosing, resulting in amore personalized treatment.

Among different imaging modalities (e.g., radiography, magneticresonance imaging and ultrasound), optical imaging holds severaladvantages. Fluorescent molecular probes are highly sensitive, possess ahigh spatial resolution, enable simultaneous multicolor imaging andspecificity, by signal activation in the tissue of interest, they maypossess high target to background ratio (TBR), and are relativelyinexpensive. Furthermore, they do not hold long term health risks, likeother commonly-used computed tomography (e.g., PET-positron emissiontomography and SPECT-single-photon emission computed tomography), whichexpose the patient to ionizing radiation.

An ideal theranostic nanomedicine system should hold (i) longcirculation time in the body, (ii) high specificity to the targettissue, (iii) an efficient release mechanism, (iv) an imaging probe thatenables monitoring its activity, (v) deep tissue penetration, and (vi)high target-to-background (TBR) ratio. High specificity can be obtainedvia passive targeting, by exploiting the enhanced permeability andretention (EPR) effect or via an additional functional targeting moiety.

In contrast to thin layer imaging of cells or surfaces, the signal fromfluorescent probes in vivo is impeded by the emitted fluorescence fromtissues and biomolecules (e.g., water, melanin, proteins andhemoglobin), which absorb photons in the wavelengths range of 200-650 nm(i.e., low signal-to-noise ratio). In addition, tissues contribute toreflection, refraction and scattering of incident photons, thusincreasing the background and blur of the obtained image. The ‘imagingwavelength window’ left for intravital imaging in order to overcomethese obstacles is at the near infra-red (NIR) range (i.e., 650-1450nm). In this range, auto-fluorescence is minimal and scattering of lightis reduced, enabling deep tissue penetration and facilitatingnon-invasive monitoring.

One way to maximize the signal from the target and to minimize thesignal from background (i.e., high TBR ratio), is the use of activatableoptical probes. The fluorescent signal is silenced/“OFF” underphysiological conditions, and is turned-ON at a designated site and/orunder specific conditions [Lee et al., Activatable molecular probes forcancer imaging. Vol. 10. 2010. 1135-44].

Although numerous classes of Turn-ON optical probes have been describedin the literature for detection of chemical and biological factors[Karton-Lifshin, N., et al., J Am Chem Soc, 2011. 133(28): p. 10960-5;Kobayashi, H., et al., Chem Rev, 2010. 110(5): p. 2620-40; Lee, S., etal., Chem Commun (Camb), 2008(36): p. 4250-60; Redy-Keisar, O., et al.,Nat Protoc, 2014. 9(1): p. 27-36; Weinstain, R., et al., Chem Commun(Camb), 2010. 46(4): p. 553-5], to this point, most polymer-basedtheranostic nanomedicines studies utilize an ‘always ON’ theranosticsystems. In these systems, a fluorescent signal is obtained from thebackground and desired site at once, resulting in low TBR.

Among methods used to obtain a selective Turn-ON mechanism, Försterresonance energy transfer (FRET) is the most common and efficient. UsingFRET technique to monitor drug release, two types of fluorophores areincorporated into the core of drug-carrying nanoparticles and serve asenergy donors and acceptors. In this process, following excitation ofthe donor, the acceptor will absorb the emission energy of the donor andwill turn off the fluorescent signal. The donor and the acceptor arerequired to have overlapping emission and absorbance spectra, as well asclose proximity between them. A FRET-based probe is turned-ON upondistance that results in the diffusion of the donor fluorophore awayfrom the acceptor, and generation of a measurable fluorescent signal[Lee et al. 2010 supra; Johansson, M. K., et al., Journal of theAmerican Chemical Society, 2002. 124(24): p. 6950-6956]. This processincludes two approaches, fluorophore-fluorophore (self-quenching) andfluorophore-quencher activation. The donor is always a fluorophore,however the acceptor can be either a quencher—a dye with no nativefluorescence (FRET) or a second fluorophore (self-quenching) [Redy, O.,et al., Org Biomol Chem, 2012. 10(4): p. 710-5].

In the fluorophore-fluorophore (self-quenching) approach, excitedfluorophores of similar type absorb the energy from each other thatwould otherwise have led to an emitted photon, thus reducing thefluorescence of the entire compound. This can occur when the excitationand emission peaks overlap or when the Stokes shift is small, like inthe case of Cy5. Hence, the fluorophore can serve as a quencher andadsorb the excitation energy. Under these circumstances the emittedenergy from one fluorophore is absorbed by another fluorophore(self-quenching) [Melancon, M. P., et al., Pharm Res, 2007. 24(6): p.1217-24].

Self-quenching involving only fluorophores may still yield weakfluorescence even in the quenched state. A second alternative tofluorophore-fluorophore quenching, is to use a fluorophore-quenchercombinations in which the quencher is non-fluorescent and plays as theacceptor, whereas the donor is a fluorophore. When a FRETfluorophore-quencher process occurs, the excited fluorophore cantransfer its emission energy to the nearby quencher [Redy, O., et al.,Org Biomol Chem, 2012. 10(4): p. 710-5].

Optical imaging in the near-infrared (NIR) range enables detection ofmolecular activity in vivo due to high penetration of NIR photonsthrough organic tissues and low auto-fluorescence background. Cyaninedyes are widely employed as fluorescence labels for NIR imaging, sincethey are compounds with large extinction coefficient and relatively highquantum yield.

In order to generate a Turn-ON system for a cyanine molecule, a FRET(fluorescence resonance energy transfer) approach is usually applied. Insuch approach, the cyanine dye and a quencher are attached through aspecific linker to obtain a quenched fluorophore. A linker, which iscleaved by a specific enzyme, separates the fluorophore from thequencher and thus, turn-ON its fluorescence signal. Exemplary suchFRET-based probes are described in Redy, O., et al., Org Biomol Chem,2012. 10(4): p. 710-5, which is incorporated by reference as if fullyset forth herein. An alternative approach, to turn OFF and ON afluorophore, could be achieved by disrupting the pull-push conjugatedπ-electron system of the dye. Such a concept, referred to as InternalCharge Transfer (ICT) probe, is described in WO 2012/123916, which isincorporated by reference as if fully set forth herein, and inKisin-Finfer E., et al., 1; 24(11):2453-8; Bioorg Med Chem Lett. 2014,which is also incorporated by reference as if fully set forth herein.

Additional background art includes Jones et al. Langmuir, 2001, 17 (9),pp 2568-2571; U.S. Patent Application Publication No. 20120122734;Theodora Krasia-Christoforou and Theoni K. Georgiou, J. Mater. Chem. B,2013, 1, 3002-3025; Morton et al., Biomaterials. 2014 April; 35(11):3489-3496; and Luk and Zhang, Appl. Mater. Interfaces 2014, 6,21859-21873.

SUMMARY OF THE INVENTION

Although polymeric nanocarriers conjugated to low molecular weight drugsgreatly improve their efficacy and toxicity profile, these nanocarrierslack information concerning drug-release time and location. Combiningtherapeutic and diagnostic modalities on the same delivery system,thereby forming theranostic (therapy and diagnostic) nanomedicine,enables simultaneous monitor and treatment of angiogenesis-dependentdiseases, like cancer. Information obtained from theranosticnanomedicines allows tuning therapy dose, while monitoring diseasedtissue and delivery kinetics. This, from a clinical prospective, mayincrease early detection of disease and decrease drug under-dosing orover-dosing, resulting in a more personalized treatment.

The present inventors have now designed various theranostic systems,which are based on a polymeric system in which a fluorogenic moiety isattached to a portion of the backbone units composing the polymericbackbone of a polymeric moiety, wherein the fluorogenic moiety isattached to the backbone units via a cleavable linking such that uponcleavage of the linking moiety, a fluorescent moiety is generated, and adetectable signal can be measured.

According to an aspect of some embodiments of the present inventionthere is provided a polymeric system comprising a first polymeric moietycomprising a polymeric backbone composed of a plurality of backboneunits and having attached to at least a portion of the backbone units afluorogenic moiety, the fluorogenic moiety being attached to thebackbone units via a first cleavable linking moiety such that uponcleavage of the linking moiety, a fluorescent signal is generated, thesystem further comprising a therapeutically active agent, such that: (i)the fluorogenic moiety is attached to one portion of the backbone unitsand the therapeutically active agent is attached to another portion ofthe backbone units; (ii) the therapeutically active agent forms a partof the fluorogenic moiety; (iii) the therapeutically active agent isattached to the first cleavable linking moiety; or (iv) the systemfurther comprises a second polymeric moiety comprising a secondpolymeric backbone composed of a plurality of backbone units and havingattached to at least a portion of the backbone units a therapeuticallyactive agent.

According to some of any of the embodiments described herein, upon thecleavage, a fluorescent moiety is generated.

According to some of any of the embodiments described herein, thefluorescent moiety emits UV-vis light.

According to some of any of the embodiments described herein, thefluorescent moiety emits near infrared light.

According to some of any of the embodiments described herein, thefluorescent moiety is or comprises a cyanine dye.

According to some of any of the embodiments described herein, the firstcleavable linking moiety is a first biocleavable linking moiety.

According to some of any of the embodiments described herein, the firstcleavable linking moiety is an enzymatically-cleavable linking moiety.

According to some of any of the embodiments described herein, the firstpolymeric moiety further comprises a quenching agent.

According to some of any of the embodiments described herein, thefluorogenic agent is attached to one portion of the backbone units andthe quenching agent is attached to another portion of the backboneunits.

According to some of any of the embodiments described herein, thequenching agent forms a part of the fluorogenic moiety.

According to some of any of the embodiments described herein, thefluorogenic moiety is represented by, or comprises a moiety representedby, formula II:

wherein:

Z₁ and Z₂ are each independently a substituted or unsubstitutedheterocylic moiety;

R₁ is hydrogen, a substituted or unsubstituted alkyl or a substituted orunsubstituted cycloalkyl;

n is an integer of from 1 to 10; and

R′ and R″ are each independently hydrogen, a substituted orunsubstituted alkyl and a substituted or unsubstituted cycloalkyl, or,alternatively, R′ and R″ form together an aryl.

According to some of any of the embodiments described herein, Z₁ and Z₂are each independently a substituted or unsubstituted heteroaryl.

According to some of any of the embodiments described herein, thefluorogenic moiety is represented by, or comprises a moiety representedby, formula IIA or IIB, as depicted herein.

According to some of any of the embodiments described herein, thefluorogenic moiety comprises a fluorescent moiety linked by the firstcleavable linking moiety or by a degradable spacer to the quenchingagent.

According to some of any of the embodiments described herein, thefluorogenic moiety is represented by a formula selected from FormulaIIIA, IIIB, IIIC, and IIID, as depicted herein.

According to some of any of the embodiments described herein, thefluorogenic moiety is represented by Formula IV, as depicted herein.

According to some of any of the embodiments described herein, thefluorogenic moiety is attached to one portion of the backbone units andthe therapeutically active agent is attached to another portion of thebackbone units.

According to some of any of the embodiments described herein, the systemfurther comprises a second polymeric moiety comprising a secondpolymeric backbone composed of a plurality of backbone units and havingattached to at least a portion of the backbone units a therapeuticallyactive agent.

According to some of any of the embodiments described herein, thetherapeutically active agent is attached to the backbone units via asecond cleavable linking moiety.

According to some of any of the embodiments described herein, the secondlinking moiety is a biocleavable linking moiety.

According to some of any of the embodiments described herein, the secondlinking moiety is an enzymatically-cleavable linking moiety.

According to some of any of the embodiments described herein, the firstand second cleavable linking moieties are the same or are cleavable bythe same mechanism (e.g., the same enzyme).

According to some of any of the embodiments described herein, thetherapeutically active agent forms a part of the fluorogenic moiety, oris attached to the first cleavable linking moiety, and wherein upon thecleavage, the therapeutically active agent is released. According tosome embodiments, upon the cleavage, a fluorescent moiety is generated.

According to some of any of the embodiments described herein, thefluorescent moiety is or comprises a cyanine dye.

According to some of any of the embodiments described herein, thetherapeutically active agent forms a part of the fluorogenic moiety, andthe fluorogenic moiety is represented by Formula VIA, VIB, VIC, or VID,as depicted herein.

According to some of any of the embodiments described herein, thetherapeutically active agent forms a part of the fluorogenic moiety, andthe fluorogenic moiety is represented by Formula IIIA, IIIB, IIIC orIIID, and wherein the therapeutically active agent is attached to one ofthe spacers or to the cleavable linking moiety.

According to some of any of the embodiments described herein, thetherapeutically active agent forms a part of the fluorogenic moiety, andthe fluorogenic moiety is represented by Formula IV, wherein thetherapeutically active is attached to the donor moiety or to thecleavable linking moiety.

According to some of any of the embodiments described herein, thebackbone units in the first polymeric backbone and/or in the secondpolymeric backbone, if present, form a polymeric backbone of HPMAco-polymer.

According to some of any of the embodiments described herein, thebackbone units in the first polymeric backbone and/or in the secondpolymeric backbone, if present, form a polymeric backbone of a PGApolymer.

According to an aspect of some embodiments of the present inventionthere is provided a polymeric conjugate comprising a polymeric backbonecomposed of a plurality of backbone units and having attached to atleast a portion of the backbone units a fluorogenic moiety, thefluorogenic moiety being attached to the portion of backbone units via acleavable linking moiety such that upon cleavage of the linking moiety,a fluorescent moiety is generated, wherein the fluorescent moiety is acyanine dye.

According to some of any of the embodiments described herein, thepolymeric conjugate further comprises a quenching agent attached to thepolymeric backbone.

According to some of any of the embodiments described herein, thefluorogenic moiety is represented by, or comprises a moiety representedby, formula II, as depicted herein.

According to some of any of the embodiments described herein, thefluorogenic moiety is represented by, or comprises a moiety representedby, formula IIA or IIB, as depicted herein.

According to some of any of the embodiments described herein, thefluorogenic moiety comprises a fluorescent moiety linked by a cleavablelinking moiety and/or a degradable spacer to a quenching agent.

According to some of any of the embodiments described herein, thefluorogenic moiety is represented by a formula selected from FormulaIIIA, IIIB, IIIC and IIID as depicted herein.

According to some of any of the embodiments described herein, thefluorogenic moiety is represented by Formula IV, as depicted herein.

According to some of any of the embodiments described herein, thepolymeric conjugate further comprises a therapeutically active agentattached to the cleavable linking moiety or forming a part of thefluorogenic moiety, such that upon the cleavage, the therapeuticallyactive agent is released.

According to an aspect of some embodiments of the present inventionthere is provided a polymeric system comprising a fluorogenic cyaninemoiety covalently attached via a cleavable linking moiety to a quenchingagent, such that upon cleavage of the linking moiety, a fluorescentcyanine moiety is generated, the system further comprising a polymericmoiety attached to the fluorogenic cyanine moiety.

According to some of any of the embodiments described herein, thepolymeric system is represented by a formula selected from Formula VA orVB, as depicted herein.

According to some of any of the embodiments described herein, thecyanine moiety is attached to the polymeric moiety via a spacer.

According to some of any of the embodiments described herein, thepolymeric system further comprises a therapeutically active agent,wherein:

(i) the therapeutically active agent is attached to the cleavablelinking moiety;

(ii) the therapeutically active agent is attached to the degradablespacer; or

(iii) the therapeutically active agent is attached to a second polymericmoiety.

According to an aspect of some embodiments of the present inventionthere is provided a polymeric system as described in any one of theembodiments described herein, where the system comprises atherapeutically active agent, for use in the treatment and diagnosis ofa medical condition treatable by the therapeutically active agent, orfor use in the preparation of a medicament for treating the medicalcondition.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a medical condition, the methodcomprising administering to a subject in need thereof a polymeric systemas described herein, which comprises a therapeutically active agent thatis usable in treating the medical condition.

According to some of any of the embodiments described herein, themedical condition is cancer.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1A-1E present the chemical structure and cleavage mechanism byCathepsin B of an exemplary HPMA copolymer-Cy5 (FIG. 1A); an exemplaryHPMA copolymer-PTX and a release mechanism of PTX therefrom by cathepsinB (FIG. 1B); an exemplary HPMA copolymer-PTX-FK and a release mechanismof PTX therefrom by cathepsin B (FIG. 1C); an exemplary HPMAcopolymer-Cy5-PTX (FIG. 1D), and an exemplary HPMA copolymer-Cy5-PTX-FK,according to some embodiments of the present invention.

FIG. 2 is a scheme depicting a two-step synthesis of HPMA-GFLG-en 10%copolymer-Cy5 conjugate, carried out by activation of Cy5 with NHSgroup, followed by coupling with HPMA copolymer.

FIG. 3 is a scheme depicting a synthesis of HPMA copolymer-PTXconjugate, carried out by activation of PTX with PNp-C1, followed byconjugation of HPMA to PTX, by mixing activated PTX with HPMA-GFLG-en 10mol % copolymer.

FIG. 4 is a scheme depicting a synthesis of HPMA copolymer-PTX-FKconjugate, carried out by forming a Phe-Lys-PABC linker and conjugatingthe linker to PTX, followed by coupling the PTX-Phe-Lys with HPMAcopolymer.

FIG. 5 is a scheme depicting a synthesis of HPMA copolymer-Cy5-PTXconjugate, carried out by conjugating of Cy5 to HPMA copolymer, andactivation of PTX with 4-Nitrophenyl, followed by its conjugation to theHPMA copolymer-Cy5 so as to generate HPMA copolymer-Cy5-PTX.

FIG. 6 is a scheme depicting a synthesis of HPMA copolymer-Cy5-PTX-FKconjugate, carried out by conjugating Cy5 to HPMA copolymer, forming aFK-PABC linker and conjugating the linker to PTX, followed by couplingthe PTX-Phe-Lys to the HPMA copolymer-Cy5-PTX-FK so as to generate HPMAcopolymer-Cy5-PTX-FK; DCM=dichloromethane, DMF=N,N-dimethylformamide,TFA=trifluoroacetic acid.

FIGS. 7A-7D present a scheme depicting a synthesis of a PGA polymer,carried out by hexylamine-initiated polymerization (FIG. 7B) of theN-carboxyanhydride (NCA) of y-benzyl-L-glutamate (FIG. 7A), followed bydeprotection (FIG. 7C); and of a PGA-PTX conjugate, carried out byactivation of PGA with CDI coupling reagent supported by DMAP as acatalyst in basic environment and conjugation of PGA to PTX, by mixingactivated polymer with PTX (FIG. 7D).

FIGS. 8A-8B present schemes depicting a synthesis of PGA-PTX-Cy5conjugate, carried out by removing the BOC protecting group of aCy5-NH₂-BOC (FIG. 8A); and conjugating the obtained Cy5-NH₂ to a PGA-PTXconjugate, by mixing an activated PGA with the Cy5-NH₂ (FIG. 8B).

FIGS. 9A-9B present graphs showing PTX release kinetics from HPMAcopolymer-PTX-FK conjugate upon incubation in the absence (diamonds) orpresence (squares) of cathepsin B [1 Unit/ml] in phosphate buffer (pH 6)(FIG. 9A); and PTX release kinetics from HPMA copolymer-PTX conjugateupon incubation in the presence of cathepsin B [1 Unit/ml] (diamonds)(FIG. 9B).

FIGS. 10A-10D present graphs showing the anti-proliferative activity ofPTX and HPMA copolymer-PTX conjugate in murine 4T1 cells (FIG. 10A) andin HUVEC cells (FIG. 10B), upon incubation for 96 hours; theanti-proliferative activity of HPMA copolymer-PTX-FK conjugate in humanMDA-MB-231 mammary adenocarcinoma cells, upon incubation for 72 hours(FIG. 10C), and the IC50 values obtained in these assays (FIG. 10D).

FIGS. 11A-11C present comparative plots showing the self-quenchingcapability of an HPMA copolymer-Cy5 conjugate (3.8 mol % loading) (blankdiamonds) and of free Cy5 (squares) (FIG. 11A); Comparative plotsshowing the changes in fluorescence intensity (λ_(Ex)=600 nm, λ_(Em)=670nm) emitted upon incubation of HPMA copolymer-Cy5 conjugate [0.01 mM] inthe presence (blank diamonds) of cathepsin B [1 Units/ml] in Phosphatebuffer (pH 6) and in the absence of cathepsin B in PBS (pH 7.4)(squares), with data acquired throughout 160 hours following enzymeaddition at 37° C. (FIG. 11B); and a bar graph showing the in vitrodegradation of HPMA copolymer-Cy5 (gray bars) in cultured MDA-MB-231cells, compared to non-treated cells (white bars), as measured byactivation of a fluorescence signal (The data represent mean SD (n=3);*p<0.05, **p<0.01) (FIG. 11C).

FIGS. 12A-12B present graphs showing quantification of the flourescencesignal following intra-tumoral injection of free Cy5 [0.1 mM; 30 μl](blank diamonds) and equivalent dose of HPMA copolymer-Cy5 conjugate(squares) into subcutaneous 4T1 mammary adenocarcinoma (FIG. 12A) andimages showing that the fluorescence signal of HPMA copolymer-Cy5conjugate is maintained 8 hours following injection, while free Cy5exhibits 80% bleach already within 3 hours [Images were acquired andquantified using CRI Maestro™ imaging system; Filter set: excitation—635nm, emission cutoff—675 nm] (FIG. 12B).

FIGS. 13A-13C present an image (FIG. 13A) and a bar graph (FIG. 13B)showing the fluorescent signal and tumor/background ratio of HPMAcopolymer-Cy5 conjugate in a 4T1 tumor, upon administering the conjugate(10 μM; 200 μl) via the tail vein of mice, as monitored using CRIMaestro™ imaging system; and a bar graph (FIG. 13C) showing theCy5-fluorescent spectrum (composed images of unmixed multispectralcubes) in resected organs of mice bearing 4T1 tumors treated with HPMAcopolymer-SQ-Cy5 conjugate (10 μM; 200 μl), demonstrating greaterintensity of Cy5-fluorescence spectrum in tumor tissue, liver andkidneys compared with other organs.

FIGS. 14A-14C present graphs showing the emitted fluorescence intensity(λ_(Ex)=650 nm) by HPMA copolymer-SQ-Cy5-PTX conjugate (FIG. 14A) andHPMA copolymer-SQ-Cy5-PTX-FK conjugate (FIG. 14B) as measured usingSpectraMax® M5^(e) plate reader, upon incubation of the conjugates [0.01mM] in the presence or absence of cathepsin B [1 Units/ml] in Phosphatebuffer (pH 6); Data was acquired throughout 48 hours following enzymeaddition at 37° C.; and comparative plots showing the emission ofHPMA-PTX-Cy5 conjugate loaded with 5.38 mol % Cy5 and HPMA-PTX-FK-Cy5loaded with 1.95 mol % Cy5 compared to free Cy5 emission (λ_(ex)=650 nm,λ_(em)=670 nm) with a similar equivalent concentration of Cy5 (13-19μM)(FIG. 14C).

FIGS. 15A-15D present comparative plots showing: the absorption spectrumof PGA-PTX-Cy5 (red) compared to a free Cy5 (blue) (FIG. 15A); theemission spectrum (λ_(ex)=650 nm, λ_(em)=670 nm) of PGA-PTX-Cy5conjugate loaded with 4 mol % Cy5 (red), PGA-PTX-Cy5 loaded with 7.5 mol% Cy5 (green) and of free Cy5 (blue) (FIG. 15B); the emittedfluorescence (λ_(ex)=650 nm, λ_(em)=670 nm) following enzymatic releaseof Cy5 from the PGA-PTX-SQ-Cy5 conjugate upon incubation in the presence(black) and in the absence (dashed gray) of cathepsin B enzyme [1Units/ml] as a function of time (FIG. 15C); and the PTX release kineticsfrom PGA-PTX-Cy5 conjugate upon incubation in the presence of cathepsinB enzyme [1 Units/ml] as a function of time (FIG. 15D).

FIGS. 16A-16D present comparative plots showing the anti-proliferativeactivity of free PTX, PGA-PTX and PGA-PTX-Cy5 conjugate, free PTX,PGA-PTX and PGA-PTX-Cy5 in human MDA-MB-231 mammary adenocarcinoma cellline (FIG. 16A), murine 4T1 cell line (FIG. 16B) and human WM239Amelanoma cell line (FIG. 16C), upon incubating the cells with the testedagent for 72 hours; and the IC50 values obtained in these assays (FIG.16D) [Data represents mean±SD. The X-axis is presented at a logarithmicscale].

FIG. 17 presents a bar graph demonstrating the inhibition of themigration of HUVECs by a PGA-PTX-Cy5 conjugate, compared to free PTX,PGA-PTX, PGA-PTX-Cy5 and control (non-treated HUVECs). Migration wasnormalized to percent migration with 100% representing migration control[Data represents mean±SD, (*** p<0.005)].

FIGS. 18A-18B present representative images showing the effect of freePTX, PGA-PTX, PGA-PTX-Cy5, PGA and Cy5-amine, compared to control(untreated), on capillary-like tube structures formation of HUVEC,following incubation (FIG. 18A); and a bar graph showing a quantitativeanalysis of the mean length of capillary tubes following incubation(Data represents mean displayed as % of control ±SD; * p<0.05; **p<0.01; *** p<0.005).

FIGS. 19A-19B present schemes depicting the Chemical syntheses ofMA-Gly-Gly-diamine-Boc monomer (FIG. 19A) and MA-Gly-Phe-Leu-Gly-PABAmonomer (FIG. 19B); NHS=N-hydroxy-succinimide, DCC=dicyclohexylcarbodiimide, DMF=N,N-dimethylformamide, NMM=N-methylmorpholine,THF=tetrahydrofuran.

FIG. 20 is a scheme depicting a two-step synthesis of HPMAcopolymer-Gly-Phe-Leu-Gly-PABC-Cy5-PTX, carried out by RAFTpolymerization of the copolymer precursor, followed by coupling withPTX, as an exemplary drug and Cy5, as an exemplary fluorogenic moiety.

FIG. 21 is a scheme depicting a two-step synthesis of HPMAcopolymer-Gly-Phe-Leu-Gly-PABC-FITC-PTX, carried out by RAFTpolymerization of the copolymer precursor, followed by coupling withPTX, as an exemplary drug, and FITC, as an exemplary fluorescent moiety.

FIGS. 22A-22B present schemes depicting the syntheses of exemplary drugand dye dipeptide-PABC moieties: Boc-NH-LG-PABC-PTX, Boc-NH-LG-PABC-Cy5and Boc-NH-LG-PABC-FITC (FIG. 22A), and ivDde-NH—FK-PABC-PTX andivDde-NH—FK-PABC-Cy5 (FIG. 22B), useful for further conjugation to HPMAcopolymer-dipeptide-ONp (Gly-Gly-ONp).

FIG. 23 presents an illustration of the general design and mode ofaction of a FRET-based turn-ON system.

FIG. 24 is a scheme depicting a two-step synthesis of HPMAcopolymer-Gly-Phe-Leu-Gly-PABC-Cy5-Quencher-PTX, carried out by RAFTpolymerization of the copolymer precursor, followed by coupling with thedrug (PTX), dye (Cy5) and finally the quencher.

FIG. 25 is a scheme depicting a two-step synthesis of HPMAcopolymer-Gly-Phe-Leu-Gly-PABC-FITC-DR1-PTX, carried out by RAFTpolymerization of the copolymer precursor, followed by coupling with thedrug (PTX), dye (FITC) and finally the quencher, DR1.

FIG. 26 is a scheme depicting a FRET-based theranostic system in which aquencher-amine is coupled to a COOH end-functionalized HPMAcopolymer-PTX-Cy5 conjugate, providing a conjugate with one quenchermolecule per polymeric chain.

FIG. 27 is a scheme depicting a FRET-based theranostic system in which aDR1-amine is coupled to a COOH end-functionalized HPMAcopolymer-PTX-FITC conjugate, providing a conjugate with one quenchermolecule per polymeric chain.

FIG. 28 is a scheme depicting the synthesis of a HPMAcopolymer-Gly-Phe-Leu-Gly-PABC-PTX-Cy5-quencher conjugate by RAFTpolymerization of a copolymer precursorHPMA-Gly-Phe-ONp/Gly-Gly-diamine-Boc, followed by coupling to theprecursor amine-Leu-Gly-PABC-PTX, amine-Leu-Gly-PABC-Cy5 andquencher-COOH.

FIG. 29 is a scheme depicting the synthesis of a HPMAcopolymer-Gly-Phe-Leu-Gly-PABC-PTX-FITC-DR1 conjugate by RAFTpolymerization of a copolymer precursorHPMA-Gly-Phe-ONp/Gly-Gly-diamine-Boc, followed by coupling to theprecursor amine-Leu-Gly-PABC-PTX, amine-Leu-Gly-PABC-FITC and DR1-amine.

FIG. 30 is a scheme depicting the synthesis of a FRET-basedPGA-Cy5-Quencher conjugate. Coupling of PGA to the Cy5-NH₂ is carriedout by mixing a CDI activated polymer and the fluorophore, followed bythe coupling of PGA-Cy5 conjugate to a deprotected Quencher-NH₂.

FIGS. 31A-31B present a scheme depicting the structure and chemicalsynthesis of a FRET-based probe-polymer conjugate based on Cy5conjugated to PEG, a latent central linker conjugated to phenyl-boronicester as a triggering substrate for hydrogen peroxide, and a quencher(FIG. 31A), and the generation of a fluorescent signal upon contact withhydrogen peroxide (FIG. 32B).

FIGS. 32A-32B present comparative plots (FIG. 32A) showing the NIRfluorescence (λ_(ex)=630 nm, λ_(em)=670 nm) emitted upon incubation ofthe Cy5-PEG conjugate [30 μM] in the presence or absence of hydrogenperoxide (5 equivalents) in 0.1 M PBS, pH 7.4, monitored by RP-HPLC;gradient: 10-90% ACN in 0.1% TFA in water; and images acquired using CRIMaestro™ Imaging system (FIG. 32B).

FIG. 33 presents an image acquired using CRI Maestro™ Imaging system(λ_(ex)=630 nm, λ_(em)=670 nm) of SCID mice bearing -U-87 MG tumors, 2minutes after injection i.v. of 200 μl of a 1 μM solution of a PEG-Cy5conjugate via the tail vein.

FIG. 34 presents the chemical structure and a schematic illustration ofthe activation mechanism of a FRET-based cathepsin B fluorescent probewith the cyanine dye Cy5.

FIG. 35 is a scheme depicting a synthetic pathway for preparing aFRET-based cathepsin B-activated fluorescent probe with the cyanine dyeCy5.

FIG. 36 presents comparative plots showing the NIR fluorescence(λ_(ex)=620 nm, λ_(em)=670 nm) emitted upon incubation of a FRET-basedcathepsin B fluorescent probe with the cyanine dye Cy5 [25 μM, 10% DMSO]in the presence (red) or absence (blue) of cathepsin B [1.4 U/ml] inactivity buffer (pH=6.0) solution.

FIGS. 37A-37B present comparative images showing the NIR fluorescenceturn-ON response of a FRET-based cathepsin B fluorescent probe with thecyanine dye Cy5 upon reaction with cathepsin B (solutions in activitybuffer of pH 6.0). FIG. 37A presents images of the probe [0.01 mM] inthe presence and in the absence of cathepsin B [10 U/ml] (1 minute afterenzyme's addition) (most and second left vials, respectively), and ofand Cy5 [0.01 mM] under the same conditions (third and fourth from theleft, respectively. FIG. 37B presents images of the probe [0.01 mM] inthe presence (4 hours after addition) and in the absence of cathepsin B[10 U/ml]. Images were taken by CRI Maestro™ Imaging system. Filter set:excitation at 635 nm, emission cut-off filter of 675 nm.

FIG. 38 presents a quantification of time-dependent fluorescence signalupon intratumoral injection of a FRET-based cathepsin B fluorescentprobe into cathepsin B-overexpressing 4T1 mammary adenocarcinoma [50 μl;0.01 mM]. Images were acquired and quantified using non-invasiveintravital CRI Maestro™ imaging system. Filter set: excitation at 635nm, emission cut-off filter of 675 nm.

FIG. 39 is a scheme depicting the synthesis of HPMAcopolymer-Gly-Gly-Phe-Lys-PABC-PTX-Cy5-Quencher by RAFT polymerizationof copolymer precursor HPMA-Gly-Gly-ONp followed by coupling toamine-Phe-Lys-PABC-PTX, amine-Phe-Lys-PABC-Cy5-Quencher as an examplefor FRET-based fluorescent Turn-On moiety.

FIG. 40 presents a schematic illustration of a general design and modeof action of an ICT-based fluorescent probe.

FIG. 41 is a scheme depicting the synthesis of HPMAcopolymer-Gly-Gly-Phe-Lys-PABC-PTX-QCy7 by RAFT polymerization ofcopolymer precursor HPMA-Gly-Gly-ONp followed by coupling toamine-Phe-Lys-PABC-PTX, amine-Phe-Lys-PABC-QCy7 as an example forICT-based fluorescent Turn-On moiety.

FIG. 42 is a schematic illustration presenting an activation mechanismof QCy7-based probe by cathepsin B to release free Camptothecin drug andproduce a fluorescent turn-ON response.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to therapyand diagnosis (theranostic) and, more particularly, but not exclusively,to polymeric systems in which a labeling agent or a labeling agent and atherapeutically active agents are attached to a polymeric backbone, toprobes useful for inclusion in such polymeric systems, and to usesthereof in diagnostic and theranostic applications.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

The present invention, in some embodiments thereof, relates to therapyand diagnosis (theranostic) and, more particularly, but not exclusively,to polymeric systems in which a labeling moiety (e.g., a fluorescent orfluorogenic moiety) or a labeling moiety and a therapeutically activeagents are attached to a polymeric backbone, and to uses thereof indiagnostic and theranostic applications.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

The present inventors have now devised and successfully practiced twotheranostic systems, which permit simultaneous drug release and imagingability: (i) a polymeric system composed of two separate polymericmoieties, one designed to release a therapeutically active agent and onedesigned to generate a fluorescent signal; and (ii) a combined polymericsystem in which a fluorogenic moiety and therapeutically active agentare attached to a single polymeric backbone.

In some embodiments, the diagnostic system is composed of an efficienthigh-loading, FRET-based (self-quenched (SQ) or paired) “Turn-ON” systemwith a NIR fluorescent cyanine dye or an analog thereof. In someembodiments, the therapeutic system includes a therapeutically activeagent, such as an anti-cancer agent (e.g., paclitaxel; PTX).

In some embodiments, the polymers are water soluble, non-toxic,biocompatible and stable polymers (e.g., HPMA, PEG or the biodegradablePGA).

In some embodiments, the cyanine dye and/or a therapeutically activeagent are conjugated to the polymeric backbone in a manner enablingtheir site-specific cleavage, for example, by a tumor-specific enzymesuch as cathepsin B.

According to an aspect of some embodiments of the present inventionthere is provided a polymeric system comprising a first polymeric moietywhich comprises a first polymeric backbone composed of a plurality ofbackbone units and having attached to at least a portion of the backboneunits a fluorogenic moiety, the fluorogenic moiety being attached to thebackbone units via a first cleavable linking moiety such that uponcleavage of the linking moiety, a fluorescent signal is generated. Thefirst polymeric moiety described herein represents the diagnostic partof a theranostic system. The first polymeric moiety described herein isa polymeric conjugate in which a fluorogenic moiety is conjugated to thefirst polymeric backbone.

According to some of any of the embodiments of the present invention,the polymeric system further comprises a therapeutically active agent.

In some embodiments, the polymeric system comprises a second polymericmoiety which comprises a second polymeric backbone composed of aplurality of backbone units and having attached to at least a portion ofthe backbone units a therapeutically active agent. This second polymericmoiety represents the therapeutic part of a theranostic system. Thesecond polymeric moiety described herein is a polymeric conjugate inwhich a therapeutically active agent is conjugated to the secondpolymeric backbone. The second polymeric backbone can be the same ordifferent from the first polymeric backbone. In some of theseembodiments, the therapeutically active agent is attached to thebackbone units via a second cleavable linking moiety, which can be thesame as or different from the first cleavable linking moiety.

In some embodiments, the therapeutically active agent is attached to aportion of the backbone units of the first polymeric backbone, such thatthe fluorogenic moiety is attached, via the cleavable linking moiety, toone portion of the backbone units, and the therapeutically active agentis attached to another portion of the backbone units. Such a polymericsystem represents a single polymeric theranostic system. In some ofthese embodiments, the therapeutically active agent is attached to thebackbone units via a second cleavable linking moiety, which can be thesame as or different from the first cleavable linking moiety. Such asystem can be regarded as a polymeric system which comprises twopolymeric moieties or polymeric conjugates, each comprising a polymericbackbone, namely, a first and a second polymeric backbone as describedherein, whereby the second polymeric backbone forms a part of the firstpolymeric backbone, resulting in a polymeric backbone in which thefluorogenic moiety is attached, via the cleavable linking moiety, to oneportion of the backbone units, and the therapeutically active agent isattached to another portion of the backbone units.

In some embodiments, the therapeutically active agent forms a part ofthe fluorogenic moiety, such that upon the cleavage of the firstcleavable linking moiety, the therapeutically active agent is releasedand a fluorescent signal is generated. Such a polymeric systemrepresents a single polymer theranostic system.

In some embodiments, the therapeutically active agent and thefluorogenic moiety are both attached to the first cleavable linkingmoiety, for example, by means of a spacer, as described herein, suchthat upon cleavage of the first cleavable linking moiety, thetherapeutically active is released and a fluorescent signal isgenerated. Such a polymeric system represents a single polymertheranostic system.

Thus, in some embodiments of the present invention the polymeric systemcan comprise two (or more) polymeric conjugates, each comprising apolymeric backbone, which can be the same or different. One of thepolymeric conjugates, referred to herein as a first polymeric moiety,comprises a fluorogenic moiety attached to a portion of the backboneunits of a first polymeric backbone. Another polymeric conjugate,referred to herein as a second polymeric moiety, comprises atherapeutically active agent attached to a portion of the backbone unitof the second polymeric backbone.

In other embodiments of the present invention, the polymeric systemcomprises one polymeric conjugate, referred to herein as a firstpolymeric moiety, in which both the fluorogenic moiety and thetherapeutically active agent are attached to the same polymericbackbone, each being attached to a portion of the backbone units in thepolymeric backbone. In these embodiments, the second polymeric backboneforms a part of the first polymeric backbone, such that the conjugatecomprises one polymeric backbone.

According to some embodiments of the invention, the first and the secondpolymeric backbones are not covalently associated therebetween, suchthat the system comprises two separate polymeric conjugates (polymericmoieties).

According to some embodiments of the invention, the second polymericbackbone forms a part of the first polymeric backbone, such that thepolymeric system comprises a polymeric backbone comprising a pluralityof backbone units having the fluorogenic moiety attached to one portionof the backbone units and having the therapeutically active agentattached to another portion of the backbone units, such that the systemcomprises one polymeric conjugate or moiety as described herein.

In some of any of the embodiments described herein, the fluorogenicmoiety is attached to the first cleavable linking moiety via a spacer.In some embodiments, the first cleavable linking moiety is attached tothe respective portion of backbone units of the first polymeric backbonevia a spacer.

In some of any of the embodiments described herein, the therapeuticallyactive agent is attached to the second cleavable linking moiety via aspacer. In some embodiments, the second cleavable linking moiety, ifpresent, is attached to the respective portion of backbone units of thepolymeric backbone via a spacer.

In some of the embodiments described herein, when the therapeuticallyactive agent forms a part of the fluorogenic moiety, the therapeuticallyactive agent is attached to the fluorogenic moiety via a spacer.

In some of any of these embodiments, the spacer is a degradable spacer,as described herein.

In some of any of the embodiments described herein, the first polymericmoiety, which comprises the fluorogenic moiety, further comprises aquenching agent, as described herein.

In some embodiments, the quenching agent is attached to a portion of thebackbone units of the first polymeric backbone, such that thefluorogenic moiety is attached to one portion of the backbone units ofthe first polymeric backbone and the quenching agent is attached toanother portion of the backbone units of the first polymeric backbone.

In some embodiments, the quenching agent is attached to a terminus ofthe first polymeric backbone, that is, the quenching agent is attachedto a terminal backbone unit of the first polymeric backbone.

The quenching agent can be attached to the backbone unit(s) via alinking moiety, or via a spacer, which can be degradable ornon-degradable.

In some embodiments, the quenching agent forms a part of the fluorogenicmoiety. In some of these embodiments, the quenching agent is attached toa fluorescent moiety via a spacer.

The polymeric conjugates described herein can be used each separately orin any combination thereof.

The polymer:

As used herein, the term “polymer” or “polymeric moiety” describes asubstance composed of a plurality of repeating structural units(backbone units) covalently connected to one another and forming apolymeric backbone. The term “polymer” as used herein encompassesorganic and inorganic polymers and further encompasses one or more of ahomopolymer, a copolymer or a mixture thereof (a blend). The term“homopolymer” as used herein describes a polymer that is made up of onetype of monomeric units and hence is composed of homogenic backboneunits. The term “copolymer” as used herein describes a polymer that ismade up of more than one type of monomeric units and hence is composedof heterogenic backbone units. The heterogenic backbone units can differfrom one another by the pendant groups thereof.

The term “polymer” or “polymeric moiety” is used herein to describe thepolymeric backbone to which the agents/moieties described herein areattached.

The polymer comprises a polymeric backbone which is comprised ofbackbone units whereby one or more of the therapeutically active and thefluorogenic moiety, and optionally other agents and/or moieties asdescribed herein, are attached to at least a portion of these backboneunits. Some or all of these backbone units are typically functionalizedprior to conjugation, so as to have a reactive group for attaching thetherapeutically active agent and/or the fluorogenic moiety and/or otheragents or moieties. Those backbone units that are not functionalizedand/or do not participate in the conjugation of the therapeuticallyactive agent and/or the fluorogenic moiety and/or other agents ormoieties, are referred to herein as “free” backbone units.

Polymers which are suitable for use in the context of the presentembodiments are biocompatible, non-immunogenic and non-toxic. Thepolymers serve as carriers that enable targeting to and delivery intotumor tissue, possibly due to the EPR effect.

The polymer may be a biostable polymer, a biodegradable polymer or acombination thereof. The term “biostable”, as used in this context ofembodiments of the invention, describes a compound or a polymer thatremains intact under physiological conditions (e.g., is not degraded invivo).

The term “biodegradable” describes a substance which can decompose underphysiological and/or environmental conditions into breakdown products.Such physiological and/or environmental conditions include, for example,hydrolysis (decomposition via hydrolytic cleavage), enzymatic catalysis(enzymatic degradation), and mechanical interactions. This termtypically refers to substances that decompose under these conditionssuch that 50 weight percents of the substance decompose within a timeperiod shorter than one year.

The term “biodegradable” as used in the context of embodiments of theinvention, also encompasses the term “bioresorbable”, which describes asubstance that decomposes under physiological conditions to break downproducts that undergo bioresorption into the host-organism, namely,become metabolites of the biochemical systems of the host-organism.

The polymer can be water-soluble or water-insoluble. In someembodiments, the polymer is water soluble at room temperature.

The polymer can further be a charged polymer or a non-charged polymer.Charged polymers can be cationic polymers, having positively chargedgroups and a positive net charge at a physiological pH; or anionicpolymers, having negatively charged groups and a negative net charge ata physiological pH. Non-charged polymers can have positively charged andnegatively charged group with a neutral net charge at physiological pH,or can be non-charged.

In some embodiments, the polymer has an average molecular weight in therange of 100 Da to 800 kDa. In some embodiments, the polymer has anaverage molecular weight lower than 60 kDa. In some embodiments, thepolymer's average molecular weight range is 15 to 40 kDa.

Polymeric substances that have a molecular weight higher than 10 kDatypically exhibit an EPR effect, as described herein, while polymericsubstances that have a molecular weight of 100 kDa and higher haverelatively long half-lives in plasma and an inefficient renal clearance.Accordingly, a molecular weight of a polymeric conjugate can bedetermined while considering the half-life in plasma, the renalclearance, and the accumulation in the tumor of the conjugate.

The molecular weight of the polymer can be controlled, at least to someextent, by the degree of polymerization (or co-polymerization).

The polymer used in the context of embodiments of the invention can be asynthetic polymer or a naturally-occurring polymer. In some embodiments,the polymer is a synthetic polymer.

The polymeric backbone of a polymeric conjugate as described herein maybe derived from, or correspond to, a polymeric backbone of polymers suchas, for example, polyacrylates, polyvinyls, polyamides, polyurethanes,polyimines, polysaccharides, polypeptides, polycarboxylates, andmixtures thereof.

Exemplary polymeric backbones which are suitable for use in the contextof the present embodiments are polymeric backbones which correspond tothe polymeric backbones of polymers such as, but are not limited to,polyglutamic acid (PGA), a poly(hydroxyalkylmethaacrylamide) (HPMA), apolylactic acid (PLA), a polylactic-co-glycolic acid (PLGA), apoly(D,L-lactide-co-glycolide) (PLA/PLGA), a polyamidoamine (PAMAM), apolyethylenimine (PEI), dextran, pollulan, a water soluble polyaminoacid, and a polyethylenglycol (PEG).

These polymers can be of any molecular weight, as described herein, andpreferably have a molecular weight within the range of 10 to 60 kDa, orof 10 to 40 kDa.

It is to be understood that the polymers as discussed herein describethose polymers that are formed from homogenic or heterogenic,non-functionalized monomeric units, and that the polymeric backboneconstituting the polymeric conjugates disclosed herein corresponds tosuch polymers by being comprised of the same monomeric units, while someof these monomeric backbone units have moieties attached thereto, asdescribed herein. Thus, the polymeric backbone of a polymeric conjugateis similar to that of the polymers described herein, and differs fromthe polymers by having the above-described agents attached to at leastsome of the backbone units therein.

In some of any of the embodiments described herein, the polymericbackbone of a polymeric moiety or conjugate corresponds to (as describedherein), or is derived from (as described herein), a polymeric backboneof a poly(hydroxyalkylmethaacrylamide) or a copolymer thereof. Such apolymeric backbone comprises methacrylamide backbone units havingattached thereto either 2-hydroxypropyl groups or such 2-hydroxypropylgroups that have been modified by attaching thereto (directly orindirectly) the moieties described herein (e.g., therapeutically activeagent(s) and/or fluorogenic moiety).

Poly(hydroxyalkylmethacrylamide) (HPMA) polymers are a class ofwater-soluble synthetic polymeric carriers that have been extensivelycharacterized as biocompatible, non-immunogenic and non-toxic. Oneadvantage of HPMA polymers over other water-soluble polymers is thatthey may be tailored through relatively simple chemical modifications,in order to regulate their respective drug and targeting moiety content.Further, the molecular weight and charge of these polymers may bemanipulated so as to allow renal clearance and excretion from the body,or to alter biodistribution while allowing tumor targeting.

In some of any of the embodiments described herein, the polymericbackbone is derived from, or corresponds to, polyglutamic acid (PGA).PGA is a polymer composed of units of naturally occurring L-glutamicacid linked together through amide bonds. The pendant free γ-carboxylgroup in each repeating unit of L-glutamic acid is negatively charged ata neutral pH, which renders the polymer water-soluble. The carboxylgroups also provide functionality for drug attachment. PGA isbiodegradable and FDA-approved.

Cysteine proteases, particularly cathepsin B, play key roles in thelysosomal degradation of PGA to its nontoxic basic components,L-glutamic acid, D-glutamic acid and D,L-glutamic acid. The cellularuptake of negatively charged polymers can be hindered due toelectrostatic repulsion forces between the polymers and the rathernegatively charged surface of the cells. Although PGA is no exception tothis rule, it does not diminish the EPR effect and the accumulation andretention of PGA-drug conjugates in solid tumors. Specificreceptor-mediated interactions of PGA-drug conjugates containingtargeting ligands may also increase the rate of polymer uptake into thetarget cells.

As used herein, “a polyglutamic acid” or “polyglutamic acid polymer”encompasses poly(L-glutamic acid), poly(D-glutamic acid),poly(D,L-glutamic acid), poly(L-gamma glutamic acid), poly(D-gammaglutamic acid) and poly(D,L-gamma glutamic acid).

PGA is usually prepared from poly(γ-benzyl-L-glutamate) by removing thebenzyl protecting group with the use of hydrogen bromide. A sequentialcopolymer of protected PGA may be synthesized by peptide couplingreactions. For the preparation of high-molecular-weight homopolymers andblock or random copolymers of protected PGA, tri-ethylamine-initiatedpolymerization of the N-carboxyanhydride (NCA) of γ-benzyl-L-glutamateis used.

Water-soluble copolymers such as N-2-hydroxypropyl methacrylamide (HPMA)copolymer and polyglutamic acid (PGA) are biocompatible, non-immunogenicand non-toxic carriers that enable specific delivery into tumor tissue(Satchi-Fainaro et al. Nat Med 2004; 10: 255-261). These macromoleculesdo not diffuse through normal blood vessels but rather accumulateselectively in the tumor site because of the EPR effect. This phenomenonof passive diffusion through the hyperpermeable neovasculature andlocalization in the tumor interstitium is observed in many solid tumorsfor macromolecular agents and lipids.

For any of the polymeric moieties or conjugates described herein, theplurality of the backbone units forming the polymeric backbone in theconjugate comprises two or more different portions of backbone unitsthat differ from one another by the presence and/or nature of the moietyor agent attached thereto. For example, one portion of the backboneunits are “free” backbone units, and one portion of the backbone unitshave a fluorogenic moiety attached thereto. In another example, a thirdportion of the backbone units have a therapeutically active agentattached thereto, or a quenching agent attached thereto.

The different backbone units that have a moiety or agent attachedthereto can be randomly dispersed within the polymeric backbone.

Thus, in some embodiments, a polymeric backbone as described herein isformed of a plurality of backbone monomeric units, which are covalentlylinked to one another so as to form the polymeric backbone. The backboneunits are therefore such that, if not having certain moieties attachedthereto, as described herein, form a polymeric backbone of a polymer.The plurality of backbone units as described herein, and the polymericbackbone comprised thereof, are therefore also defined herein as derivedfrom, or corresponding to, the polymeric backbone of such a polymer. Theplurality of backbone units as described herein, and the polymericbackbone comprised thereof, therefore correspond to, or are derivedfrom, a polymer, whereby one or more moieties or agents, as describedherein, are attached to one or more portions of the backbone units.Since once the one or more moieties are attached to one or more portionsof the backbone units forming the polymeric backbone, the backbone unitsforming the polymeric backbone are not identical to one another, as isthe case of an “intact” polymer, and hence the polymeric conjugate isactually a copolymer, or has a copolymeric backbone, which is comprisedof two or more types of backbone units. The phrase “polymeric backbone”as used herein therefore describes a “copolymeric backbone” comprised ofat least two different types of backbone units.

It is to be noted that portions of the backbone units differ from oneanother by the presence and type of the moiety or agent that areattached to the backbone unit, but maintain the chemical structure ofthe portion of the backbone unit that forms the polymeric backbone. Inanalogy to a peptide, where the portions of the backbone units differfrom one another by the side chain of the amino acid, the portions ofthe backbone units differ from one another by the presence and/or natureof the pendant group thereof.

In some of any of the embodiments described herein, a polymericconjugate or moiety as described herein comprises a polymeric (orcopolymeric) backbone formed from a plurality of backbone units, and theplurality of backbone units comprise one or more of the followingbackbone units:

-A₁-, which represents a backbone unit within the polymeric backbone,or, in other words, a backbone unit of the polymer from which thepolymeric backbone is derived, and is “free” of moieties that attachedthereto;

-A₂-, which represents a backbone unit of the polymer from which thepolymeric backbone is derived (a backbone unit within the polymericbackbone), having a fluorogenic moiety (F), as described herein,attached thereto via a cleavable linking moiety, as described in furtherdetail hereinafter;

-A₃-, which represents a backbone unit of the polymer from which thepolymeric backbone is derived (a backbone unit within the polymericbackbone), having a therapeutically active agent (D), as describedherein attached thereto, optionally via a cleavable linking moiety, asdescribed in further detail hereinafter;

-A₄-, which represents a backbone unit of the polymer from which thepolymeric backbone is derived (a backbone unit within the polymericbackbone), having a quenching agent attached thereto; and optionally

-A₅-, which represents a backbone unit of the polymer from which thepolymeric backbone is derived (a backbone unit within the polymericbackbone), having a functional/reactive group attached thereto. Suchbackbone units can be present in a polymeric moiety as described herein,in cases where a polymer comprising a plurality of functionalizedbackbone units is used for forming a polymeric conjugate as describedherein, whereby not all the functionalized backbone units participate inthe conjugation reaction to form one or more of A₂, A₃ or A₄ asdescribed herein. Such backbone units can be regarded as “free” backboneunits to the extent that they do not contain a moiety or agent asdescribed herein conjugated thereto, yet they contain areactive/functional pendant group, which is denoted herein as R.

The backbone units can be arranged within the polymeric backbone in anyorder, such that each of the backbone units can be a terminal backboneunit, which is attached to one other backbone unit, or is attached totwo other backbone units, which can be the same or different.

In some of any of the embodiments of the present invention, a polymericmoiety or conjugate as described herein comprises at least backboneunits A₂ as described herein, and optionally also backbone units A₁, andfurther optionally also backbone units A₄ and A₅. Such a polymericmoiety represents a diagnostic part of a theranostic system, and in someembodiments, a polymeric system comprising such a polymeric moiety,further comprises a second polymeric moiety.

In some of these embodiments, the second polymeric moiety comprisesbackbone units A₃ as described herein, and optionally also backboneunits A₁, and further optionally also backbone units A₅.

In some of any of the embodiments of the present invention, a polymericmoiety or conjugate as described herein comprises backbone units A₁, A₂and A₃ as described herein, and optionally also backbone units A₄ andA₅.

In some of any of the embodiments of the present invention, the moietyor agent attached to the backbone units can be conjugated or attacheddirectly to pendant group of the backbone units, or indirectly, via aspacer or a linker, as described herein.

In some embodiments, the plurality of backbone units forming thepolymeric backbone comprises the following portions of backbone units:

-(A₁)w-;

-(A₂-F)x-;

-(A₃-D)y-; and

-(A₄-Q)s,

and optionally -(A₅-R)z

wherein:

A₁ is a backbone unit within the polymeric backbone, as describedherein;

A₂-F is a backbone unit within the polymeric backbone having attachedthereto, via a cleavable linking moiety, a fluorogenic moiety F, asdescribed herein;

A₃-D is a backbone unit within the polymeric backbone having attachedthereto a therapeutically active agent D, as described herein;

A₄-Q is a backbone unit within the polymeric backbone having attachedthereto a quenching agent (Q), as described herein;

and A₅ in a functionalized backbone unit within the polymeric backbone,as described herein, wherein R is said reactive or functional group.

The backbone units can further comprise second and third linkingmoieties, and/or spacers, through which the agents or moieties areattached, as described in further detail hereinafter.

Herein, the phrases “loading onto the polymer”, or simply “load”, areused to describe the amount of an agent or moiety that is attached tothe polymeric backbone of the conjugates described herein, and isrepresented herein by the mol percent (mol %) of the backbone unitshaving the agent or moiety attached thereto, as defined hereinafter.

Herein “mol percent” represents the number of moles of backbone unitshaving the agent or moiety attached thereto, as defined hereinafter, per1 mol of the polymeric backbone, multiplied by 100, and hence representsthe number of moles of an attached moiety or agent per 1 mol of thepolymer, multiplied by 100.

The % loading can be measured by methods well known by those skilled inthe art, some of which are described hereinbelow under the Materials andMethods of the Examples section that follows.

The mol percent of each of the backbone units is represented herein by“w”, “x”, y”, “s”, and “z”, respectively. “x”, “y” and “s” represent theloading of the respective moieties.

In some of any of the embodiments described herein, a load of atherapeutically active agent, when present within the polymeric moiety,denoted herein also as “y”, ranges from 0.1 to 100 mol percent, or from0.1 to 20 mol percent, and can be, for example, 0.1, 0.5, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20, and evenhigher values, including any subranges and values therebetween.

In some of any of the embodiments described herein, the load of thefluorogenic moiety, denoted also as “x” herein, ranges from 0.1 to 100mol percent, or from 0.1 to 20 mol percent, or from 1 to 20 mol percent,and can be, for example, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19 and 20, and even higher values, includingany subranges and values therebetween.

In some of any of the embodiments described herein, the load of thequenching agent, if present within separate backbone units, denoted alsoas “s” herein, ranges from 0.1 to 50 mol percent, or from 0.1 to 20 molpercent, or from 1 to 20 mol percent, and can be, for example, 0.1, 0.5,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and20, and higher values, including any subranges and values therebetween.

According to some embodiments of the invention, w is an integer having avalue such that x/(x+y+w+s+z) multiplied by 100 is in the range of from70 to 99.9; y is an integer having a value such that y/(x+y+w+s+z)multiplied by 100 is in the range of from 0.01 to 20, as describedherein; and x is an integer having a value such that w/(x+y+w+z+x)multiplied by 100 is in the range of from 0.01 to 20, as describedherein.

For example w/(x+y+w+z+x) multiplied by 100 may be 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99 or 99.9; y/(x+y+w+z+s) multiplied by 100 may be0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; x/(x+y+w+z+s)multiplied by 100 may be 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14 or 15; s/(x+y+w+z+s) multiplied by 100 may be 0.01, 0.02, 0.03, 0.04,0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14 or 15.

It would be appreciated that x, y, s, z and w can be controlled asdesired by selecting the mol ratio of the respective monomeric unitsused for forming the polymeric conjugate, as discussed hereinbelow.

Any of the polymeric systems described herein, in some embodiments, havea large enough hydrodynamic diameter. The term “large enough” is usedherein to describe a polymeric moiety having a hydrodynamic diameterwhich leads to an increase in the ratio of polymer accumulated in tumortissue as compared to other tissues. The determination of the optimalratio is well within the capability of those skilled in the art. Forexample, the ratio may be 1.1, 2, 3, 4, 5 etc. In some embodiments, thehydrodynamic diameter is in the range of from 15 nm to 200 nm. In someembodiments, the hydrodynamic diameter is in the range of from 50 nm to150 nm. In some embodiments the hydrodynamic diameter is in the range offrom 70 nm to 90 nm. In yet another embodiment the hydrodynamic diameteris 95 nm. The hydrodynamic diameter can be measured by methods known inthe art.

The polymeric moieties described hereinabove may be administered orotherwise utilized in this and other aspects of the present invention,either as is, or as a pharmaceutically acceptable salt, enantiomer,diastereomer, solvate, hydrate or a prodrug thereof.

The phrase “pharmaceutically acceptable salt” refers to a chargedspecies of the parent compound and its counter ion, which is typicallyused to modify the solubility characteristics of the parent compoundand/or to reduce any significant irritation to an organism by the parentcompound, while not abrogating the biological activity and properties ofthe administered compound. The neutral forms of the compounds arepreferably regenerated by contacting the salt with a base or acid andisolating the parent compound in a conventional manner. The parent formof the compound differs from the various salt forms in certain physicalproperties, such as solubility in polar solvents, but otherwise thesalts are equivalent to the parent form of the compound for the purposesof the present invention.

The phrase “pharmaceutically acceptable salts” is meant to encompasssalts of the moieties and/or polymeric backbone which are prepared withrelatively nontoxic acids or bases, depending on the particularsubstituents found on the compounds described herein. When polymericmoieties of the present embodiments contain relatively acidicfunctionalities, base addition salts can be obtained by contacting theneutral (i.e., non-ionized) form of such conjugates with a sufficientamount of the desired base, either neat or in a suitable inert solvent.Examples of pharmaceutically acceptable base addition salts includesodium, potassium, calcium, ammonium, organic amino, or magnesium salt,or a similar salt. When polymeric moieties of the present inventioncontain relatively basic functionalities, acid addition salts can beobtained by contacting the neutral form of such polymeric moieties witha sufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike (see, for example, Berge et al., “Pharmaceutical Salts”, Journal ofPharmaceutical Science, 1977, 66, 1-19). Polymeric moieties of thepresent embodiments may contain both basic and acidic functionalitiesthat allow the conjugates to be converted into either base or acidaddition salts.

The neutral forms of the polymeric moieties are preferably regeneratedby contacting the salt with a base or acid and isolating the parentpolymeric moiety in a conventional manner. The parent form of thepolymeric moiety differs from the various salt forms in certain physicalproperties, such as solubility in polar solvents, but otherwise thesalts are equivalent to the parent form of the polymeric moiety for thepurposes of the present invention.

The Fluorogenic Moiety:

As generally stated in the art, the term “fluorogenic” encompasses astate or condition of having the capability to be fluorescent (i.e. toabsorb and emit light, as defined hereinbelow) following a chemical,biochemical or physical occurrence or event. Thus, a “fluorogenicmoiety” or a “fluorogenic compound” describes a non-fluorescent moietyor compound or a weakly fluorescent moiety or compound that becomes morefluorescent (e.g., by at least 10%, at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 905, at least 100% (at least 2-fold), optionally at least 3-fold,optionally at least 4-fold more fluorescent, and optionally at 10-foldor higher more fluorescent) upon the occurrence of a chemical,biochemical or physical event.

As used herein, a “chemical event” describes an event that involves achange in the chemical structure of a compound, including, but notlimited to, bond cleavage, bond formation, protonation, deprotonation,oxidation, reduction, and more.

In some of any of the embodiments described herein, the chemical eventis bond cleavage.

The phrase “fluorogenic moiety” as used herein therefore describes amoiety which changes its fluorescence upon a chemical event, and istherefore regarded, and in also referred to herein interchangeably, as achemically-activatable fluorogenic moiety, as a probe, as achemically-activatable probe or as a Turn-ON probe. The fluorogenicmoieties described herein throughout are also referred to in the contextof the fluorescent (dye) compounds or moieties generated upon saidactivation.

The phrase “Turn-ON” is used herein to describe a fluorogenic moiety,which upon a chemical event, becomes fluorescent, as defined herein. Inthe fluorogenic moiety, fluorescence is OFF, yet, upon being subjectedto a chemical event, fluorescence turns ON (and a fluorescent signal isgenerated). The chemical event comprises a cleavage of a linking moiety,as described herein.

The phrase “fluorescent” refers to a compound or moiety that emits lightupon return to the base state from a singlet excitation. The fluorescentcompounds or moieties disclosed herein are also referred to hereinthroughout as dye compounds or dye molecules or as fluorophores or asfluorchromes.

In some embodiments, the emitted light is a near infrared light (near IRor NIR), being in the range of from about 700 nm to about 1400 nm. Insome embodiments, the emitted light has emission maxima at a wavelengththat is suitable for biological applications (e.g., in vivoapplications), which ranges from 650 nm to 900 nm, and in someembodiments, from 700 nm to 800 nm.

Thus, a fluorogenic moiety as disclosed herein does not exhibitfluorescence and hence does not emit light at a near infrared range, forexample, a light having a wavelength in the range of from about 650 nmto about 900 nm, and is designed such that it is capable of exhibitingfluorescence and thus of emitting light at such a near infrared rangeupon said cleavage.

In some embodiments, the emitted light is a UV-vis light. Thus, afluorogenic moiety as disclosed herein does not exhibit fluorescence andhence does not emit light at a UV-vis range, and is designed such thatit is capable of exhibiting fluorescence and thus of emitting light atsuch a UV-vis range upon said cleavage.

In some of any of the embodiments described herein, a fluorogenic moietycomprises a fluorescent moiety or compound, which is attached to saidcleavable linking moiety (optionally via a spacer such as a degradablespacer), yet, the fluorescence of the moiety is quenched and hence isOFF. Once the linking moiety is cleaved, and the fluorescent moiety orcompound is released from the polymeric moiety, and diffuses awaytherefrom, quenching is no longer effected, the fluorescence of themoiety turns ON, and a fluorescent signal is generated. A polymericmoiety or system comprising such a fluorogenic moiety is also referredto herein as a FRET system, or FRET-based system, as is discussed indetail hereinafter.

In some of these embodiments, the quenching of the fluorescence isself-quenching (SQ), and is effected by having at least two fluorescentmoieties per a polymeric backbone. Once the linking moiety is cleaved,and the fluorescent moieties or compounds are released from thepolymeric moiety, and diffuse away therefrom, self-quenching is nolonger effected, the fluorescence of the moieties turns ON, and afluorescent signal is generated.

In some of these embodiments, the quenching of the fluorecsence iseffected by means of a quenching agent.

In some embodiments, the quenching agent is attached to one or morebackbone units of the first polymeric backbone, and the fluorescentmoiety is attached to other, one or more, backbone units of thepolymeric backbone units, such that the fluorescence of the fluorescentmoiety is quenched and is OFF. Once the linking moiety is cleaved, andthe fluorescent moiety or compound is released from the polymericmoiety, and diffuses away therefrom, quenching is no longer effected,the fluorescence of the moiety turns ON, and a fluorescent signal isgenerated.

In other embodiments, the quenching agent forms a part of thefluorogenic moiety. In these embodiments, the fluorogenic moietycomprises a quenching agent attached to a fluorescent moiety, optionallyvia a spacer such as a degradable spacer, such that upon the cleavage ofthe first linking moiety, the fluorescent moiety is released, quenchingis no longer effected, the fluorescence of the fluorescent moiety turnsON and a fluorescent signal is generated.

In some of any of the embodiments described herein, the fluorogenicmoiety comprises a compound or a moiety which is non-fluorescent (i.e.,does not absorb and emit light) or which has weak fluorescence (e.g.,have a quantum yield lower by at least 2-fold than a quantum yield of acorresponding fluorescent molecule with the strong fluorescence), whenattached to the polymeric backbone, and which becomes more fluorescent,as defined herein, upon a chemical event, due a change (rearrangement)in the structure of the compound or moiety. In some of theseembodiments, the change in the structure of the compound or moietyinvolves relocalization of π-electrons, as a result of the chemicalevent (cleavage of the linking moiety), which may generate, for example,a conjugated π-electron system, which accounts for fluorescence. Apolymeric moiety or system comprising such a fluorogenic moiety is alsoreferred to herein as an ICT system, or ICT-based system, as isdiscussed in detail hereinafter.

In some of any of the embodiments described herein, the fluorogenicmoiety has a cyanine-like structure and the fluorescent moiety orcompound is a cyanine dye or a cyanine-like dye.

As used herein, the phrase “cyanine-like structure” describes a moleculethat has two nitrogen-containing moieties which are joined by apolymethine-containing chain (e.g., a carbomethine chain). One or bothnitrogens can be a part of a nitrogen-containing heteroaromatic moiety,or, alternatively, be a secondary or tertiary ammonium.

In some embodiments, the polymethine-containing chain comprises 2 carbonatoms and the cyanine-like structure is of a Cy2 type cyanine structure,as this term is widely recognized in the art.

In some embodiments, the carbomethine-containing chain comprises 3carbon atoms and the cyanine-like structure is of a Cy3 type cyaninestructure.

In some embodiments, the carbomethine-containing chain comprises 5carbon atoms and the cyanine-like structure is of a Cy5 type cyaninestructure.

In some embodiments, the carbomethine-containing chain comprises 7carbon atoms and the cyanine-like structure is of a Cy7 type cyaninestructure.

In some embodiments, the carbomethine-containing chain comprises 5 or 7carbon atoms.

Thus, in some embodiments, the fluorogenic moiety as described herein isa modified cyanine dye, including any of the known cyanine dyes, whichis modified by the means used to attach it to the polymeric backbone.

In some embodiments, a cyanine-like fluorogenic moiety as describedherein can be regarded as comprising the same basic chemical arrangementas cyanine dyes, yet, because of its attachment to the polymericbackbone in sufficient amount and in close proximity, the fluorogenicmoiety is spectroscopically silenced in the NIR range before activationby said cleavage, due to self-quenching.

In some embodiments, a cyanine-like fluorogenic moiety as describedherein can be regarded as comprising the same basic chemical arrangementas cyanine dyes, yet, because of its attachment to the polymericbackbone in close proximity to a quenching agent, the fluorogenic moietyis spectroscopically silenced in the NIR range before activation by saidcleavage, due to quenching.

In some embodiments, a cyanine-like fluorogenic moiety as describedherein has a chemical arrangement which is different from cyanine dyes(e.g., a delocalized π-electrons system), and hence the fluorogenicmoiety is spectroscopically silenced in the NIR range before activationby said cleavage.

Fluorogenic moieties which have a modified cyanine structure are alsoreferred to herein as cyanine-based fluorogenic moieties, and thefluorescent moieties or compounds generated upon said cleavage arereferred to herein as cyanine dyes. Exemplary such moieties aredescribed in further detail hereinafter.

Other fluorogenic or fluorescent moieties are also contemplated.

In embodiments of the present invention where the fluorogenic moietycomprises a fluorescent moiety or compound attached to the cleavablelinker or moiety, the fluorescent moiety can be selected from the myriadof known fluorescent moieties. FITC is a non-limiting example. Otherexamples are listed in “Methods in Molecular Biology, vol. 335:Fluorescent Energy Transfer Nucleic Acid Probes: Designs and Protocols”,Edited by: V. V. Didenko © Humana Press Inc., Totowa, N.J., Chapter 2,page 17, by Mary Katherine Johans son, which is incorporated byreference as if fully set forth herein.

The Quenching Agent:

Herein, the phrase “quenching agent” is also referred to interchangeablyas “quencher”, and describes a moiety or compound which is capable ofdecreasing the fluorescence intensity of another moiety or compound, asdescribed herein. The decrease in fluorescence intensity by quenchingcan result from processes such as excited state reactions, energytransfer, complex-formation and the like.

A quencher is selected in accordance with a selected fluorescent moietyor compound, namely, as capable of, for example, absorbing energyemitted from the fluorescent moiety or compound, interacting with thefluorescent moiety or compound which it is in its excited state, etc.

In some of any of the embodiments described herein, the quencher isselected suitable for FRET, which is based on dipole-dipole interactionsbetween the transition dipoles of the fluorescent moiety (which acts asa donor) and the quencher (which acts as an acceptor). A suitablequencher should be positioned at a distance of distances up to 100 Åfrom the donor, should have a suitable orientation of the dipole momentrelative to the donor, and should have a spectral overlap with thedonor.

Those skilled in the art would readily recognize which quenching agentor dye is suitable for use for quenching the fluorescence of a selectedfluorescent moiety or moiety.

Exemplary quenching agent-fluorescent moiety pairs include, but are notlimited to, two cyanine dyes (which can be the same, for SQ, ordifferent, for pair FRET) (NIR), FITC and DR1 (visible), Fluorescein &Cy5 (visible), Cy5 & IR783 (NIR), Cy7 & IR783 (NIR), Cy3 & BHQ2(visible), Cy5 & BHQ2 (NIR). Additional pairs are listed in “Methods inMolecular Biology, vol. 335: Fluorescent Energy Transfer Nucleic AcidProbes: Designs and Protocols”, Edited by: V. V. Didenko © Humana PressInc., Totowa, N.J., Chapter 2, page 17, by Mary Katherine Johansson,which is incorporated by reference as if fully set forth herein.

The Linking Moiety:

Herein throughout, the phrase “linking moiety” is also referred toherein as “linker”.

In any of the embodiments described herein, the fluorogenic moiety isattached to the polymeric backbone (to at least a portion of thebackbone units composing the first polymeric backbone), via a linkingmoiety, which is a cleavable linking moiety (referred to herein as afirst cleavable linking moiety).

In some of any of the embodiments described herein, the therapeuticallyactive agent is attached to a polymeric backbone (e.g., to at least aportion of the first or second polymeric backbone) via a linking moiety,preferably, a cleavable linking moiety, referred to herein as a secondcleavable linking moiety. The linker linking the therapeutically activeagent to the polymeric backbone, if present, and the linker linking thefluorogenic moiety to the polymeric backbone may be the same ordifferent. In some embodiments, the linker is the same and in someembodiments, the linker or either the same or is cleavable under thesame conditions (e.g., by the same enzyme).

The linker described herein refers to a chemical moiety that serves tocouple the fluorogenic moiety and/or the therapeutically active agent tothe polymeric backbone (to the respective portion of backbone units).

The phrase “cleavable linking moiety” or “cleavable linker” describes achemical moiety which can undergo a bond cleavage upon a chemical event,as described herein.

In some embodiments, the linker is a biodegradable or biocleavablelinker.

The phrases “biodegradable linker” and “biocleavable linker” as usedherein, describe a linker that is capable of being degraded, or cleaved(undergo bond cleavage), when exposed to certain physiologicalconditions. Such physiological conditions can be, for example, pH, apresence of a certain enzyme, a presence of chemical substance (e.g.,analyte) and the like.

In some embodiments, the linker is designed as being cleavable atconditions characterizing the desired bodily site (e.g., by certainenzymes, chemical substances or pH), as detailed hereinbelow.

According to some embodiments, the biodegradable linker is apH-sensitive linker, a hydrolysable linker or an enzymatically-cleavablelinker.

In some embodiments, the linker is capable of being cleaved bypre-selected cellular enzymes, for instance, those found in osteoblasts,osteoclasts, lysosomes of cancerous cells or proliferating endothelialcells, or in tumor tissues.

Alternatively, an acid hydrolysable linker could comprise an ester oramide linkage.

In some embodiments the biodegradable linker is an enzymaticallycleavable linker. Such a linker is typically designed so as to include achemical moiety, typically, but not exclusively, an amino acid sequencethat is recognized by a pre-selected enzyme. Such an amino acid sequenceis often referred to in the art as a “recognition motif”. A polymericconjugate comprising such a linker typically remains substantiallyintact in the absence of the pre-selected enzyme in its environment, andhence does not cleave or degrade so as to the release the moietyattached thereto via the linker until contacted with the enzyme.

In some embodiments, the enzymatically cleavable linker is cleaved by anenzyme which is overexpressed in tumor tissues. A polymeric conjugatecomprising such a linker ensures, for example, that a substantial amountof a conjugated moiety is released from the conjugate only at the tumortissue.

Exemplary such enzymes include, but are not limited to, Cathepsins(cysteine proteases) such as Cathepsin B, Cathepsin K, Cathepsin D,Cathepsin H, Cathepsin L, and Cathepsin S, legumain, matrixmetalloproteinases such as MMP-2 and MMP-9, as well as MMP1, MMP3, MMP7,MMP13 and MMP14, KLK6 (kallikrein-related peptidase-6 which encodes atrypsin-like serine peptidase), PIM serine/threonine kinases such as PIM1, PIM 2 and PIM 3, histone deacetylases (HDAC) such as HDAC1, HDAC2,HDAC3, HDAC6 AND kdac8.

Suitable linking moieties having a Cathepsin K cleavable site includeamino acid sequences such as, but not limited to, -[Asn-Glu-Val-Ala]-and -[Lys-Lys]-.

Suitable linking moieties having cathepsin-B cleavable sites includeamino acid sequences such as, but are not limited to, -[Gly-Phe-Lys]-,-[Cit-Val]-, -[Arg]-, -[Arg-Arg]-, -[Val-Arg]-, -[Phe-Lys]-,-[Phe-Arg]-, [Gly-Phe-Leu-Gly], -[Gly-Phe-Ala-Leu]- and-[Ala-Leu-Ala-Leu]-, -[Gly-Leu-Gly]-, -[Gly-Phe-Gly]-,-[Gly-Phe-Leu-Gly-Phe-Lys]-, -[(Glu)₆-(Asp)₈]- and combinations thereof.

In some embodiments the linking moiety comprises the amino acidsequences -[Gly-Phe-Lys]-, -[Gly-Leu-Gly]-, -[Gly-Phe-Gly]-,-[Gly-Leu-Phe-Gly]-, -[Gly-Phe-Leu-Gly]-, -[Phe-Lys]- and-[Gly-Phe-Leu-Gly-Phe-Lys]-. In some embodiments, the trigger unitconsists of these amino acid sequences or a combination thereof.Suitable linking moieties having cathepsin-D cleavable sites include anamino acid sequence such as, but are not limited to,-[Gly-Pro-Ile-Cys(Et)-Phe-Phe-Arg-Leu]-.

Suitable linking moieties having cathepsin-K cleavable sites include anamino acid sequence such as, but are not limited to,-[Gly-Gly-Pro-Nle]-.

Suitable linking moieties having cathepsin-L cleavable sites include anamino acid sequence such as, but are not limited to, -[Phe-Arg]-.

Suitable linking moieties having Legumain cleavable sites include anamino acid sequence such as, but are not limited to, -[Ala-Ala-Asn]-,-[Asn-Glu-Val-Ala]- and -[(Glu)₆-(Asp)₈]-, and any combination thereof.

Suitable linking moieties having MMP cleavable sites include an aminoacid sequence such as, but are not limited to, -[Cys-Gly-Leu-Asp-Asp]-,-[Gly-Pro-Leu-Gly-Val]-, -[Gly-Pro-Leu-Gly-Ala-Gly]-,-[Cys-Asp-Gly-Arg]-, -[Gly-Pro-Leu-Gly-Val-Arg-Gly-Cys]- and-[Pro-Leu-Gly-Met-Thr-Ser]-, and any combination thereof. In someembodiments, the linking moieties have only a part of theabove-described amino acid sequences. In some embodiments, the linkingmoiety consists of 3 amino acids of the above-described sequences.

Suitable linking moieties having MMP-2 and MMP-9 cleavable sites includean amino acid sequence such as, but are not limited to,-[Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln]-,-[Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln]-,-[Gly-Pro-Gln-Gly-Ile-Trp-Gly-Gln]-, -[Pro-Leu-Gly-Val-Arg]-,[Pro-Leu-Gly-Leu-Tyr-Leu]-, -[Pro-Leu-Gly-Leu-Tyr-Ala-Leu]-,-[Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln]-,-[Gly-Pro-Leu-Gly-Leu-Trp-Ala-Gln]-,-[Gly-Pro-Leu-Gly-Val-Arg-Gly-Lys]-,-[His-Pro-Val-Gly-Leu-Leu-Ala-Arg]-,-[Gly-Gly-Pro-Leu-Gly-Leu-Trp-Ala-Gly-Gly]-,-[Ala-Ala-Ala-Pro-Leu-Gly-Leu-Trp-Ala]- and combinations thereof. Insome embodiments, the linking moieties have only a part of theabove-described amino acid sequences. In some embodiments, the linkingmoiety consists of 3 amino acids of the above-described sequences.

Suitable linking moieties having MMP-7 cleavable sites include an aminoacid sequence such as, but are not limited to,-[Gly-Val-Pro-Leu-Ser-Leu-Thr-Met-Gly-Cys]- and-[Arg-Pro-Leu-Ala-Leu-Trp-Arg-Ser]- and combinations thereof. In someembodiments, the linking moieties have only a part of theabove-described amino acid sequences. In some embodiments, the linkingmoiety consists of 3 amino acids of the above-described sequences.

Suitable linking moieties having MMP-13 cleavable sites include an aminoacid sequence such as, but are not limited to,-[Gly-Pro-Leu-Gly-Met-Arg-Gly-Leu-Gly-Lys]-. In some embodiments, thelinking moieties have only a part of the above-described amino acidsequence. In some embodiments, the linking moiety consists of 3 aminoacids of the above-described sequence.

Suitable linking moieties having KLK6 cleavable sites include amino acidsequences such as, but are not limited to, -[Gly-Ala-Arg-Arg-Arg-Gly]-,-[Trp-Ala-Arg-Arg-Ser]-, -[Trp-Ala-Arg-Lys -Arg]-, -[Leu-Arg-Lys-Arg-Trp]-, -[Ala-Lys -Arg-Arg-Gly]-, abd -[Trp-Lys-Lys-Lys-Arg]. Insome embodiments, the linking moieties have only a part of theabove-described amino acid sequences. In some embodiments, the linkingmoiety consists of 3 amino acids of the above-described sequences.Suitable linking moieties having PIM cleavable sites include an aminoacid sequence such as, but are not limited to,-[(Arg/Lys)₃-AA₁-[Ser/Thr-AA₂]-, with AA₁ and AA₂ being independentlyany amino acid residue except basic or large hydrophobic residues. Anexemplary amino acid sequence include:-[Ala-Arg-Lys-Arg-Arg-Arg-His-Pro-Ser-Gly-Pro-Pro-Thr-Ala]-.

Suitable linking moieties having HDAC cleavable sites include acetylatedLysine.

Suitable linking moieties having caspase cleavable sites include anamino acid sequence such as, but not limited to, -[Asn-Glu-Val-Ala]-,-[(Glu)₆-(Asp)₈]-, -[Asp-Glu-Val-Asp]-, and[Asp-Glu-Val-Asp-Ala-Pro-Lys]-.

In some embodiments, the linker is a Cathepsin B-cleavable linker.

Cathepsin B is a lysosomal enzyme over-expressed in both epithelial andendothelial tumor cells. Suitable exemplary linkers having cathepsin-Bcleavable sites include amino acid sequences such as, but are notlimited to, -[Gly-Phe-Leu-Gly]-, -[Gly-Phe-Ala-Leu]-,-[Ala-Leu-Ala-Leu]-, -[Gly-Leu-Gly]-, -[Gly-Phe-Gly]-,-[Gly-Phe-Leu-Gly-Phe-Lys]-, -[Cit-Val]-, -[Arg]-, -[Arg-Arg]-,-[Phe-Lys]-, -[Val-Arg]-, -[Phe-Arg]-, -[6-Glu-8-Asp]-, and anycombination thereof.

In some embodiments the enzymatically cleavable linker is cleaved bycathepsin K.

Cathepsin K is a lysosomal cysteine protease involved in bone remodelingand resorption and is predominantly expressed in osteoclasts. Itsexpression is stimulated by inflammatory cytokines that are releasedafter tissue injury and in bone neoplasms [Pan et al. 2006, J DrugTarget 14:425-435; Husmann et al. 2008, Mol Carcinog 47: 66-73; Segal etal. PUS One 2009, 4(4):e5233].

A non-limiting example of a linker having cathepsin K cleavable sites is-[Gly-Gly-Pro-Nle]-.

In some embodiments the linker comprises the amino acid sequences-[Gly-Leu-Gly]-, -[Gly-Phe-Gly]-, -[Gly-Leu-Phe-Gly]-,-[Gly-Phe-Leu-Gly]-, -[Phe-Lys]-, -[Gly-Phe-Leu-Gly-Phe-Lys]- and-[Gly-Gly-Pro-Nle]-. In some embodiments, the linker consists of theseamino acid sequences or a combination thereof.

Other suitable linkers include, but are not limited to, alkyl chains;alkyl chains optionally substituted with one or more substituents and inwhich one or more carbon atoms are optionally interrupted by nitrogen,oxygen and/or sulfur heteroatom. Other suitable linkers include aminoacids and/or oligopeptides.

Such alkyl chains and/or oligopeptides can optionally be functionalizedso as allow their covalent binding to the moieties linked thereby (e.g.,the polymeric backbone units and the fluorogenic moiety, the polymericbackbone units and the therapeutically active agent). Such afunctionalization may include incorporation or generation of reactivegroups that participate in such covalent bindings, as detailedhereinunder.

In some embodiment, the linker is a biodegradable oligopeptide whichcontains, for example, from 2 to 10 amino acid residues.

An oligopeptide linker which contains the pre-selected amino acidsequence (recognition motif) can also be constructed such that therecognition motif is repeated several times within the linker, tothereby enhance the selective release of the attached agent or moiety.Various recognition motifs of the same or different enzymes can also beincorporated within the linker.

Similarly, the linker may comprise multiple pH sensitive bonds ormoieties. Linkers comprising such multiple cleavable sites can enhancethe selective release of the therapeutically active agent at the desiredbodily site, thereby reducing adverse side effects, and further enhancethe relative concentration of the released drug at the bodily site whenit exhibits its activity.

In some embodiments of the present invention, the fluorogenic moietyand/or the therapeutically active agent(s) is/are bound directly to thepolymeric backbone units, whereby the bond linking these moieties to thepolymeric backbone is biodegradable, for example, a hydrolysable bond,an enzymatically-cleavable bond or a pH-sensitive bond. Such a bond canbe formed upon functionalizing the polymeric backbone units, thefluorogenic moiety and/or the therapeutically active agent, so as toinclude compatible reactive groups, as defined herein, for forming therequired bond. Such a bond is also referred to herein as a cleavable orbiocleavable linker.

According to some embodiments, the biodegradable linker is apH-sensitive linker or an enzymatically-cleavable linker.

A pH-sensitive linker comprises a chemical moiety that is cleaved ordegraded only when subjected to a certain pH condition, such as acidicpH (e.g., lower than 7), neutral pH (6.5-7) or basic pH (higher than 7).

Such a linker may, for example, be designed to undergo hydrolysis underacidic or basic conditions, and thus, the conjugate remains intact anddoes not release the agents or moieties attached to the polymericbackbone in the body, until it reaches a physiological environment wherea pH is either acidic or basic, respectively.

Exemplary pH-sensitive linkers include, but are not limited to, ahydrazone bond, ester (including orthoester) bond, amide bond ofcis-aconytil residue, a trityl group, acetals, ketals, Gly-ester and a-[Gly-Phe-Gly]- moiety.

The peptide linker may also include a peptide sequence which serves toincrease the length of the linker. Longer peptides may be advantageousdue to a more efficient steric interaction of the linker with thecleaving enzyme due to enhanced accessibility.

In some embodiments, the linker is -[Gly-Phe-Leu-Gly-Phe-Lys]-. Such alinker comprises two “recognition motifs” of Cathepsin B, and a cleavagethereof so as to release the moiety attached thereto is effected only inthe presence of high enzyme concentration. This feature enhances theselective release of the attached moiety at a site where the enzyme isover-expressed.

In some embodiments, the linker is -[Gly-Phe-Leu-Gly]-.

In some embodiments, the linker is or comprises -[Phe-Lys]-.

In some of any of the embodiments described herein, the first and secondcleavable linking moieties, if present, are the same or are cleavable bythe same chemical event.

A spacer:

In some of any of the embodiments described herein, the fluorogenicmoiety is attached to the first cleavable linker via a spacer.

In some of any of the embodiments described herein the first cleavablelinker is attached to the respective backbone units via a spacer.

In some of any of the embodiments described herein, the therapeuticallyactive agent is linked to the respective polymeric backbone units and/orto the second cleavable linker via a spacer. The spacers can be the sameor different.

In some of the embodiments described herein, the quenching agent, ifpresent, is linked to the respective polymeric backbone units via aspacer.

In some of the embodiments described herein, when the quenching agentforms a part of the fluorogenic moiety, it is linked to the fluorescentmoiety via a spacer.

In some of the embodiments described herein, when the therapeuticallyactive agent forms a part of the fluorogenic moiety, it is linked to thefluorescent moiety via a spacer.

The term “spacer” as used herein describes a chemical moiety that iscovalently attached to, and interposed between, two other moieties, or amoiety/agent and a polymeric backbone unit, thereby forming abridge-like.

In some cases, a spacer is utilized for enabling a more efficient andsimpler attachment of the fluorogenic moiety and/or therapeuticallyactive agent and/or quenching agent to the polymeric backbone units orlinker or to one another, in terms of steric considerations (e.g.,renders the site of the polymeric backbone to which coupling is effectedless hindered) or chemical reactivity considerations (adds a compatiblereactive group to facilitate coupling). In some cases, the spacer maycontribute to the performance of the resulting polymeric conjugate. Forexample, the spacer may render an enzymatically cleavable linker lesssterically hindered and hence more susceptible to enzymaticinteractions.

In some embodiments, the spacer facilitates the attachment of the moietyor agent to the polymeric backbone units or the linker. This may beeffected by imparting a reactive group to the moiety to be attached,which is chemically compatible with functional groups in the polymericbackbone units and/or the linker attached to the polymeric backbone,and/or by modifying the solubility of the moiety to be attached to thepolymer, so as to facilitate the reaction between the polymer (orco-polymer) and the moiety.

Suitable spacers include, but are not limited to, alkylene chains,optionally substituted by one or more substituents and which areoptionally interrupted by one or more nitrogen, oxygen and/or sulfurheteroatom.

Other suitable spacers include amino acids and amino acid sequences,optionally functionalized with one or more reactive groups for beingcoupled to the respective portion or moiety of the polymeric conjugate.

In some embodiments, a spacer has the formula G-(CH₂)n-K, wherein n isan integer from 1 to 10; and G and K are each a reactive group such as,for example, NH, O or S. In some embodiments, G and K are each NH and nis 2.

An exemplary spacer is —[NH—(CH₂)_(m)NH₂]— wherein “m” stands for aninteger ranging from 1-10. Preferably m is 2.

In some embodiments, the spacer is or comprises an amino acid sequence,optionally an inert amino acid sequence (namely, does not affect theaffinity or selectivity of the polymeric conjugate). Such a spacer canbe utilized for elongating or functionalizing the linker.

Exemplary such sequences include, for example, -[Gly-Gly-].

In some embodiments, the spacer is a degradable spacer, which is capableof undergoing degradation reactions so as to release an agent attachedthereto. In some embodiments, the spacer is biodegradable, as definedherein.

In some embodiments the spacer is a substituted or unsubstituted arylgroup and substituted or unsubstituted heteroaryl group whereby thesubstituents can be carbonate, C-amido, N-amido and amine, whereby thespacer may be linked to the desired agent or moiety or to the polymericbackbone units either directly, through the aromatic group oralternatively, via one or more of the substituents.

In some embodiments, the spacer is a degradable spacer selected suchthat it undergoes a spontaneous degradation once it is cleaved from thepolymeric conjugate. Such spacers are also referred to herein asself-immolative spacers.

Such a spacer can be, for example, attached to a biodegradable linker atone end and to another moiety or agent (e.g., the fluorogenic moiety) atanother end, such that once the biodegradable linker is cleaved, so asto release the spacer and the moiety attached thereto, the spacerundergoes a spontaneous degradation so as to release the moiety attachedthereto.

Exemplary spacers that can undergo such a spontaneous degradationinclude, but are not limited, chemical moieties that can undergo aspontaneous 1,4-, 1,6-, 1,8-, etc. elimination, via a cascade ofimmolative electronic reactions. Such chemical groups are known in theart, or, otherwise, can be devised by those skilled in the art.

In an exemplary embodiment, the spacer is such that can undergo aspontaneous 1,6-benzyl elimination. An example of such a spacer isp-aminobenzyl carbonate (PABC).

In some embodiments, the spacer comprises one or more of the exemplaryspacers described herein.

In some embodiments, a spacer is used to connect 3 moieties to oneanother, or to connect 2 moieties to a cleavable linking moiety or torespective polymeric backbones.

For example, a spacer can connect a fluorescent moiety and a quenchingagent and/or a therapeutically active agent to one another, so as toform a fluorogenic moiety, and to connect the fluorogenic moiety to acleavable linking moiety. Such a spacer can include a bi- ortri-functional moiety (e.g., an aryl moiety as described herein), whichis also referred to herein as a branching spacer. Such a spacer cancombine also spacers which are attached to the branching spacer, andconnect the moieties/agents to the branching spacer unit.

Exemplary such multi-functional spacers are shown, for example, in FIGS.31A-B, 34, 35, 39 and 42.

The spacer may be varied in length and in composition. A spacer asdefined herein encompasses also any combination of the spacers describedherein.

The following describes exemplary polymeric conjugates and polymericsystems comprising same according to some embodiments of the presentinvention.

The First Polymeric Moiety:

According to an aspect of some embodiments of the present inventionthere is provided a polymeric conjugate, also referred to herein as afirst polymeric moiety or a first polymeric conjugate, which comprises a(first) polymeric backbone composed of a plurality of backbone units andhaving attached to at least a portion of the backbone units afluorogenic moiety, as described herein, the fluorogenic moiety beingattached to the portion of backbone units via a cleavable linking moietysuch that upon cleavage of the linking moiety, a fluorescent signal isgenerated (e.g., upon release and/or generation of a fluorescentmoiety).

According to some embodiments of this aspect of the invention, thefluorescent moiety emits near infrared light.

According to some embodiments of this aspect of the invention, thefluorescent moiety is a cyanine dye.

According to some of any of the embodiments of the invention, the firstcleavable linking moiety is a first biocleavable linking moiety, asdescribed herein.

In some of any of the embodiments described herein, the fluorogenicmoiety is such that when it is attached to the polymeric backbone viathe first cleavable linking moiety, it does not emit light, whereby whenthe first linking moiety is cleaved, the generated fluorescent moietyemits light (e.g., near infrared light), thus featuring a Turn-ONmechanism, as described herein.

According to some of any of the embodiments described herein, thefluorogenic moiety is attached to the cleavable linking moiety via aspacer, as described in any of the respective embodiments.

According to some embodiments, the spacer is selected degradable suchthat it allows releasing or generating the fluorescent moiety, asdescribed herein, upon cleavage of the linking moiety.

According to some embodiments, the spacer is selected degradable suchthat it allows generating the fluorescent signal upon cleavage of thelinking moiety.

Spacers usable in the context of a fluorogenic moiety can be selected toact via ICT or FRET mechanism, as described herein.

In some of any of the embodiments described herein, the first polymericmoiety further comprises a quenching agent, either attached to one ormore polymeric backbone units of the first polymeric backbone or forminga part of the fluorogenic moiety, as described herein.

In some of any of the embodiments described herein, the fluorogenicmoiety is a fluorescent moiety, which is attached directly or via aspacer (e.g., a degradable or self-immolative spacer as describedherein) to the cleavable linking moiety.

In some of any of the embodiments described herein, the first polymericmoiety is a homo-FRET system, and is devoid of a quenching agent.

In some of these embodiments, a loading of the fluorescent moiety issuch that allows self-quenching, namely, the loading results in adistance between the fluorescent moieties attached to the backbone unitswhich is up to 100 angstroms.

In some of these embodiments, the loading of the fluorescent moiety isat least 1 mol %, preferably at least 2 mol %, at least 3 mol % or atleast 4 mol % and/or ranges from 1 to 10 mol %.

In some of any of the embodiments described herein, the first polymericmoiety is a pair-FRET system, which further comprises a quenching agent,as described herein.

In some of these embodiments, the quenching agent is attached to therespective backbone units via a spacer, for example, a non-degradablespacer, such as, for a non-limiting example, a spacer that comprises a-[Gly-Gly]- moiety.

In some of any of the embodiments described herein, the quenching agentis attached to a terminal backbone unit of the polymeric backbone. Insome of these embodiments, the polymeric backbone is functionalized oris designed so as to feature a reactive group, or a spacer featuring areactive group, for attaching the quenching agent. An exemplary suchgroup can be generated while synthesizing a HPMA copolymer by RAFTpolymerization, as exemplified in the Examples section that follows.

A first polymeric moiety as described herein can be represented byFormula IA:

wherein:

A₁, A₂ and A₄ are polymeric backbone units as described herein;

L₂ is the first cleavable lining moiety, as described herein,

S₂ is a first spacer, linking the fluorogenic moiety to L₂, as describedherein, or is absent;

L₄ is a third linking moiety, linking the quenching agent to thebackbone units, and which can be cleavable or non-cleavable, or isabsent;

S₄ is a third spacer linking the quenching agent to the linking moiety,or is absent;

F is a fluorogenic moiety as described in any one of the respectiveembodiments herein;

Q is a quenching agent as described in any one of the respectiveembodiments herein;

w is an integer having a value such that w/(x+s+w) multiplied by 100 isin the range of from 0 to 99.9;

x is an integer having a value such that x/(x+s+w) multiplied by 100 isin the range of from 0.1 to 100; and

s is an integer having a value such that s/(x+s+w) multiplied by 100 isin the range of from 0 to 99.9.

Each [A₂-L₂-S₂-F] independently represents a backbone unit havingattached thereto the fluorogenic moiety; and

Each [A₄-L₄-S₄-Q] independently represents a backbone unit havingattached thereto the quenching agent.

Optionally, the polymeric moiety further comprises backbone units-[A₅]z- as described herein, wherein z is an integer having a value suchthat z/(x+s+w+z) multiplied by 100 is in the range of from 0 to 99.9.

When z is other than 0, w, x and s in Formula I are divided by “x+w+z+s”instead of “x+s+w”.

In some of any of the embodiments described herein:

-   -   w is an integer having a value such that w/(x+s+w) multiplied by        100 is in the range of from 0.1 to 99.9, or from 10 to 99.9, or        from 20 to 99.9, or from 30 to 999, or from 40 to 99.9, or from        50 to 99.9, or from 60 to 99.9, or from 70 to 99.9, or as        further described herein;    -   x is an integer having a value such that x/(x+s+w) multiplied by        100 is in the range of from 0.1 to 99.9, or from 0.1 to 20, or        from 0.1 to 15, or from 1 to 15, or from 2 to 15, or from 3 to        15 or from 4 to 15, as is further described herein; and    -   s is an integer having a value such that s/(x+s+w) multiplied by        100 is in the range of from 0 to 100, or from 0 to 20, or from 0        to 15, or from 0 to 1, as is further described herein.

In some embodiments, s is 0, and the polymeric moiety is a homo-FRETsystem, as described herein.

In some embodiments, s is a positive integer and is such that a singlemolecule of a quenching agent is attached to the polymeric backbone. Insome of these embodiments, A₄ is a terminal backbone unit in thepolymeric backbone.

In some embodiments, s is a positive integer and is in a ratio to xwhich is in a range of from 20:1 to 1:20, or from 10:1:10, or from 5:1to 1:5, or from 2:1 to 1:2, or is 1:1, including any subratiostherebetween.

In some of these embodiments, each of the backbone units A₁, A₂ and A₄can be a terminal unit, attached to one other unit, or is attached totwo other units, which can be the same of different.

In some of any of the embodiments described herein, the fluorogenicmoiety is, or comprises a fluorescent moiety, as described herein.

The fluorescent moiety is also referred to herein as F*.

In some of any of the embodiments described herein, the fluorogenicmoiety is, or comprises a fluorescent moiety which is, or comprises, acyanine dye, or a cyanine-like moiety, as described herein.

In cyanine-like dye molecules, one nitrogen is positively charged (e.g.,in a form of an ammonium ion) and one nitrogen atom is neutral (e.g., ina form of an amine) and thus has a lone pair of electrons. The positivecharge in cyanine-like dyes therefore resonates between the two nitrogenatoms via the polymethine chain.

In some of these embodiments, the fluorogenic moiety is represented by,or comprises, a moiety represented by, formula II:

wherein:

Z₁ and Z₂ are each independently a substituted or unsubstitutedheterocylic moiety;

R₁ is hydrogen, a substituted or unsubstituted alkyl or a substituted orunsubstituted cycloalkyl;

n is an integer of from 1 to 10; and

R′ and R″ are each independently hydrogen, a substituted orunsubstituted alkyl and a substituted or unsubstituted cycloalkyl, or,alternatively, R′ and R″ form together an aryl.

In cyanine-like fluorescent moiety, two heterocylic moieties are linkedtherebetween via a substituted or unsubstituted polymethine chain, suchthat one heterocylic moiety acts as a donor moiety (Z₂) and one acts asan acceptor moiety (Z₁).

The phrase “polymethine chain” describes a chain of methine groups(e.g., —CH═CH— groups) each can independently be substituted, as long asthe substituent does not interfere with the optical properties of thedisclosed moiety, as defined herein.

The polymethine-containing moiety forms a chain that can comprise from 2to 13 carbon atoms, preferably from 2 to 7 carbon atoms.

Exemplary heterocyclic moieties suitable for inclusion in thefluorogenic compounds disclosed herein as donor moieties include, butare not limited to, imidazoline ring, imidazole ring, benzimidazolering, α-naphthoimidazole ring, β-naphthoimidazole ring, indole ring,isoindole ring, indolenine ring, isoindolenine ring, benzindoleninering, pyridinoindolenine ring, oxazoline ring, oxazole ring, isoxazolering, benzoxazole ring, pyridinooxazole ring, α-naphthoxazole ring,β-naphthoxazole ring, selenazoline ring, selenazole ring,benzoselenazole ring, α-naphthselenazole ring, β-naphthselenazole ring,thiazoline ring, thiazole ring, isothiazole ring, benzothiazole ring,α-naphthothiazole ring, β-naphthothiazole ring, tellulazoline ring,tellulazole ring, benzotellulazole ring, α-naphthotellulazole ring,β-naphthotellulazole ring, isoquinoline ring, isopyrrole ring,imidaquinoxaline ring, indandione ring, indazole ring, indoline ring,oxadiazole ring, carbazole ring, xanthene ring, quinazoline ring,quinoxaline ring, thiodiazole ring, thiooxazolidone ring, tetrazolering, triazine ring, naphthyridine ring, piperazine ring, pyrazine ring,pyrazole ring, pyrazoline ring, pyrazolidine ring, pyrozolone ring,pyridine ring, pyridazine ring, pyrimidine ring, pyrylium ring,pyrrolidine ring, pyrroline ring, pyrrole ring, phenazine ring,phenanthridine ring, phthalazine ring, furazan ring, benzoxazine ring,morpholine ring, and rhodanine ring.

Acceptor moieties can be an ammonium form of any of the foregoing.

In some embodiments, Z₁ and Z₂ are each independently a substituted orunsubstituted heteroaryl, whereby the acceptor moiety is positivelycharged.

In some embodiments, one of acceptor and donor moieties comprises apyridine ring and the other is an indolenine ring, as defined herein.

In some embodiments, the donor moiety is an indolenine ring, and theacceptor moiety is an ammonium form thereof.

The phrase “indolenine ring” describes a ring having an aromatic portionhaving fused thereto a 5-membered aromatic.

In some embodiments, the fluorogenic moiety is, or comprises, a moietyrepresented by Formula IIA:

wherein:

Y₁ and Y₂ are each independently a substituted or unsubstituted aromaticmoiety; and

X₁ and X₂ are each independently selected from the group consisting ofCR₃R₄, NR₃, and S, wherein R₃ and R₄ are each independently selectedfrom the group consisting of hydrogen, alkyl, cycloalkyl, aryl,heteroalicyclic, heteroaryl, alkoxy, hydroxy, thiohydroxy, thioalkoxy,aryloxy, thioaryloxy, amino, nitro, halo, trihalomethyl, cyano, amide,carboxy, sulfonyl, sulfoxy, sulfinyl, sulfonamide, and a saccharide.

Whenever the carbon, nitrogen or sulfur representing X in the aboveformula, are substituted, the substituents can be independently analkyl, cycloalkyl, alkyl, cycloalkyl, aryl, heteroalicyclic, heteroaryl,alkoxy, hydroxy, thiohydroxy, thioalkoxy, aryloxy, thioaryloxy, amino,nitro, halo, trihalomethyl, cyano, amide, carboxy, sulfonyl, sulfoxy,sulfinyl, sulfonamide, and a saccharide.

In some embodiments, Y₁ and Y₂ are each a substituted or unsubstitutedphenyl.

In some embodiments, X₁ and X₂ are each independently CR₃R₄.

In some embodiments, R₃ and R₄ are each an alkyl.

In some embodiments, R₁ is hydrogen or alkyl.

In some embodiments, the fluorogenic moiety is represented by, orcomprises, a moiety represented by, Formula IIB:

wherein:

g and k are each independently an integer of from 0 to 5; and

R₂ and R₃ are each independently selected from the group consisting ofhydrogen, alkyl, cycloalkyl, aryl, heteroalicyclic, heteroaryl, alkoxy,hydroxy, thiohydroxy, thioalkoxy, aryloxy, thioaryloxy, amino, nitro,halo, trihalomethyl, cyano, amide, carboxy, sulfonyl, sulfoxy, sulfinyl,sulfonamide, and a saccharide.

In some embodiments, R₁ is hydrogen or a substituted or unsubstitutedalkyl. In some embodiments, one or both R₂ and R₃ is an alkylsubstituted by a sulfonyl or sulfinyl.

It is to be noted that fluorogenic compounds in which one or more of theindolenine-like rings is replaced by any of the acceptor or donormoieties described herein, for example, any of the ammonium acceptormoieties described herein (e.g., a pyridinium moiety), are alsocontemplated.

In some of any of the embodiments described herein, the quenching agentforms a part of the fluorogenic moiety.

In some of these embodiments, s is 0.

In some embodiments, a FRET-based modular system is used as afluorogenic moiety, in which both the fluorescent moiety and thequenching agent are linked to one another, and/or to the first cleavablelinking moiety.

In some embodiments, such a system can be generally represented,according to some embodiments of the invention, by formulae III or III*:

wherein F* is a fluorescent moiety, as described herein; Q is thequenching agent;

S′ and S′″ (if present) are each independently a spacer, preferably oneor more of which is degradable, or absent; and

S″ is a multifunctional spacer, as described herein, for example, aself-immolative spacer, which connects the fluorogenic moiety to thefirst cleavable moiety, or to an additional spacer which is connected tothe cleavable linking moiety.

In some embodiments, at least S″ in Formula III is a degradable spacer,e.g., a self-immolative spacer, as described herein, which, uponcleavage of the linking moiety, degrades so as to no longer have thequenching agent linked to the fluorescent moiety. As a result, afluorescent signal is generated.

In some embodiments, at least S″ and S′ in Formula III* is a degradablespacer, e.g., a self-immolative spacer, as described herein, which, uponcleavage of the linking moiety, degrades so as to no longer have thefluorescent moiety linked to the quenching agent and to the polymericmoiety. As a result, a fluorescent signal is generated.

In some of any of the embodiments described herein, the fluorescentmoiety is a cyanine-like structure or moiety, as described herein, andthe fluorogenic moiety can represented by one or more of the followingformulae:

wherein:

Z₁, Z₂, R′, R″ and n are as described herein for Formula II, and canform any of the cyanine structures depicted and described herein inFormulae IIA and IIB; and

R₁ and R₂, if present, are each independently hydrogen, a substituted orunsubstituted alkyl or a substituted or unsubstituted cycloalkyl;

In some embodiments, Z₁ and Z₂ are each independently a substituted orunsubstituted heterocylic moiety, as described herein.

Exemplary such system is presented in FIG. 39.

Herein throughout, the curled line indicates an attachment point to thefirst cleavable linking moiety, either directly, or via spacer (e.g.,degradable spacer, as described herein).

In some of any of the embodiments described herein, an ICT modularsystem is used as a fluorogenic moiety. In these embodiments, thefluorogenic moiety is a modified fluorescent moiety which exhibitsreduced fluorescence, as described herein, due to an alteration in itschemical structure. Upon cleavage of the linking moiety, the fluorogenicundergoes rearrangement and a fluorescent moiety is generated. Thus, afluorescent signal is generated.

In some of these embodiments, s is 0.

In some of these embodiments, a cyanine-like fluorogenic moiety asdescribed herein has a chemical arrangement which is different fromcyanine dyes (e.g., a delocalized π-electrons system), and hence thefluorogenic moiety is spectroscopically silenced in the NIR range beforeactivation by said cleavage.

In some embodiments, such a fluorogenic moiety is represented by FormulaIV:

wherein:

Z₁ and Z₂ are each independently a substituted or unsubstitutedheterocylic moiety, as described in any one of the embodiments hereinfor an acceptor moiety of a cyanine-like structure;

R₁ and R₂ are each independently hydrogen, a substituted orunsubstituted alkyl or a substituted or unsubstituted cycloalkyl;

m and n are each independently an integer of from 0 to 4;

R′ and R″ are each independently hydrogen, a substituted orunsubstituted alkyl and a substituted or unsubstituted cycloalkyl, or,alternatively, R′ and R″ form together an aryl, as described herein; and

A is a donor-containing moiety, which, when attached to A₂ via saidfirst cleavable linking moiety, and optionally also via a spacer (e.g.,a degradable spacer) interferes with a conjugation of π electronsbetween Z₁ and Z₂, and upon cleavage, participates in said conjugationof π electrons.

The curled line denotes an attachment point.

As shown in Formula IV, both Z₁ and Z₂ moieties are acceptor moieties,and the donor moiety A is inactivated by its linkage to the linkingmoiety. Thus, the conjugation of π electrons in the moiety is disrupted.Once the linking moiety is cleaved, the donor-containing moietyfunctions as a donor, and a conjugated π electron system is generated,resulting in a fluorescent moiety and generation of a fluorescentsignal.

Unlike cyanine dyes, in the modified cyanine structures disclosedherein, the presence of a donor moiety interferes with the resonance(the delocalization of π electrons) between the two nitrogen atoms, andboth nitrogen atoms are positively charged (e.g., in a form of anammonium ion). As such, there is no delocalization of π-electrons (noresonating electrons) between the nitrogen-containing moieties.

Thus, in some embodiments, the fluorogenic moiety disclosed herein has acyanine-like structure, modified so as to include two positively chargednitrogen (e.g., ammonium)-containing moieties (instead of twonitrogen-containing moieties with one positive charge resonatingtherebetween) and a donor moiety that forms a conjugated π-electronsystem with the two ammonium-containing moieties, whereby thedonor-moiety interferes with the delocalization of the π-electronssystem of a non-modified cyanine-like molecule, by restrictingdelocalization of π-electrons to portions of the molecule that do notinvolve the nitrogen-containing moieties, and thus reduces or abolishesthe delocalization of the positive charge that is present innon-modified cyanine-like molecules.

Such a fluorogenic moiety is designed such that upon the cleavage of thefirst linking moiety, delocalization of the positive charge is restored.

Thus, the fluorogenic moieties described in these embodiments follow adesign in which the inclusion of the donor moiety results indelocalization of π electrons through a smaller portion of the molecule(smaller number of overlapping p-orbitals), as compared to non-modifiedcyanine structures, and hence the moiety is incapable of interactingwith light so as to emit NIR light.

The fluorogenic moiety disclosed herein, however, further follows adesign in which upon the cleavage of the linking moiety, rearrangementof the donor moiety occurs and results in a structure in which πelectrons are relocalized such that one of the ammonium-containingmoieties becomes an amine-containing moiety, and thus a resonatingpositive charge between two nitrogen-containing moieties, as in cyaninedyes, is restored. The π electrons relocalization thus results in amoiety that has spectroscopic behavior similar to cyanine dyes, and isthus capable of emitting NIR light.

Accordingly, the cyanine-based fluorogenic moieties described in theseembodiments are designed after known cyanine dyes, by having twonitrogen-containing moieties and a carbomethine-containing chain linkingtherebetween, yet differ from cyanine dyes by the presence of twopositively charged (e.g., ammonium) nitrogen-containing moieties(instead of one positively charged nitrogen-containing moiety), andfurther by the presence of a donor moiety as described herein.Fluorogenic moieties which are equivalent to such fluorogenic moieties,but in which the donor-containing moiety is attached to cleavable moietyY are disclosed in WO 2012/123916. Each of these moieties can be used inthese embodiments, upon the modification explained herein.

An exemplary such a system is presented in FIG. 41.

Any of the fluorogenic moieties described herein can be attached to aterminal or non-terminal backbone unit of the first polymeric backbone.

In some of any of the embodiments described herein, the backbone unitsform a polymeric backbone of a HPMA copolymer, as described herein.

In some of these embodiments, the polymeric moiety can be represented byFormula IA:

wherein the variables are as defined herein, and R represents a reactivegroup, as described herein.

In some of these embodiments, x and s are other than 0.

In some of these embodiments, the backbone units containing thequenching agent is a terminal backbone unit, and the quenching agent Qis attached to the backbone unit via a spacer (S₄).

In some of any of the embodiments described herein, the backbone unitsform a polymeric backbone of a PGA polymer, as described herein.

In some of these embodiments, the polymeric moiety can be represented byFormula IB:

wherein the variables are as defined herein.

In some of these embodiments, x and s are other than 0.

In some of any of the embodiments described herein, the backbone unitsform a polymeric backbone of a PEG polymer, as described herein. In someof these embodiments, the quenching agent forms a part of thefluorogenic moiety, and the fluorogenic moiety is attached to a terminalbackbone unit of the polymer.

A Second Polymeric Moiety:

According to some of any of the embodiments described herein, thefluorogenic moiety is attached to one portion of the backbone units andthe therapeutically active agent is attached to another portion of thebackbone units.

According to some of any of the embodiments described herein, the systemfurther comprises a second polymeric moiety comprising a secondpolymeric backbone composed of a plurality of backbone units and havingattached to at least a portion of the backbone units a therapeuticallyactive agent.

According to some of any of the embodiments described herein, thetherapeutically active agent is attached to the backbone units via asecond cleavable linking moiety.

According to some of any of the embodiments described herein, the secondlinking moiety is a biocleavable linking moiety.

According to some of any of the embodiments described herein, the secondlinking moiety is an enzymatically-cleavable linking moiety.

According to some of any of the embodiments described herein, the firstand second cleavable linking moieties are the same or are cleavable bythe same mechanism (e.g., the same enzyme).

In some embodiments, the therapeutically active is attached to thebackbone units or to the linking moiety, if present, via a spacer, asdescribed in any one of the respective embodiments.

In some embodiments, the backbone units in the second polymeric backboneform a HPMA copolymer backbone.

In some embodiments, the backbone units in the second polymeric backboneform a PGA polymer backbone.

The backbone units if the second polymeric backbone can be the same ordifferent from the backbone units of first polymeric backbone, and arepreferably the same.

A System with a Single Polymeric Backbone:

According to some of any of the embodiments described herein, thetherapeutically active agent forms a part of the fluorogenic moiety, oris attached to the first cleavable linking moiety, such that upon thecleavage of the first linking moiety, as described herein, thetherapeutically active agent is released. According to some embodiments,upon the cleavage, a fluorescent moiety is generated, as describedherein.

According to some of any of the embodiments described herein, thefluorescent moiety is or comprises a cyanine dye, as described herein.

According to some of any of the embodiments described herein, thetherapeutically active agent is attached to the first linking moiety,preferably via a degradable spacer, as described herein.

According to some of any of the embodiments described herein, thetherapeutically active agent forms a part of the fluorogenic moiety, andthe fluorogenic moiety is represented by Formula VIA, VIB, VIC, or VID,as depicted herein.

wherein:

Z₁ and Z₂ are each independently a substituted or unsubstitutedheterocylic moiety, as described herein;

R₁ and R₂ are each independently hydrogen, a substituted orunsubstituted alkyl or a substituted or unsubstituted cycloalkyl;

n is an integer of from 1 to 10;

R′ and R″ are each independently hydrogen, a substituted orunsubstituted alkyl and a substituted or unsubstituted cycloalkyl, or,alternatively, R′ and R″ form together an aryl;

S′, S″ and S′ are each independently a degradable spacer, or absent, asdescribed herein for Formulae IIIA, IIIB, IIIC and IIID; and

D is the therapeutically active agent,

wherein the curled line indicates an attachment point.

According to some of any of the embodiments described herein, both thetherapeutically active agent and the quenching agent form a part of thefluorogenic moiety, and the fluorogenic moiety is represented by FormulaIIIA, IIIB, IIIC or IIID, and wherein the therapeutically active agentis attached to one of the spacers shown therein or to the cleavablelinking moiety.

According to some of any of the embodiments described herein, thetherapeutically active agent forms a part of the fluorogenic moiety, andthe fluorogenic moiety is represented by Formula IV, wherein thetherapeutically active is attached to the donor moiety or to thecleavable linking moiety.

A polymeric system according to some of these embodiments can berepresented by Formula I:

wherein:

D is a therapeutically active agent, as described herein;

F is a fluorogenic moiety as described in any one of its respectiveembodiments; Q is a quenching agent, as described in any one of itsrespective embodiments;

L₂ is said first linking moiety;

L₃ is said second linking moiety or absent;

L₄ is a linking moiety linking the quenching agent, as described herein,or absent;

each of S₂, S₃ and S₄ is independently a spacer, as described in any oneof its respective embodiments, or absent;

w is an integer having a value such that w/(x+y+w+s) multiplied by 100is in the range of from 0 to 99.9;

x is an integer having a value such that x/(x+y+w+s) multiplied by 100is in the range of from 0.1 to 100;

y is an integer having a value such that y/(x+y+w+s) multiplied by 100is in the range of from 0 to 100; and

s is an integer having a value such that s/(x+y+w+s) multiplied by 100is in the range of from 0 to 99.9.

A₁, A₂, A₃ and A₄ are each backbone units covalently linked to oneanother and forming a polymeric backbone,

such that each [A₃-L₃-S₃-D] independently represents a backbone unithaving attached thereto said therapeutically active agent;

each [A₂-L₂-S₂-F] independently represents a backbone unit havingattached thereto said fluorogenic moiety; and

each [A₄-L₄-S₄-Q] independently represents a backbone unit havingattached thereto said quenching agent.

According to some embodiments, A₄ is a terminal backbone unit, asdescribed herein.

According to some embodiments, the quenching agent forms a part of thefluorogenic moiety, as described herein, in which case, “s” is 0.

According to some embodiments, the therapeutically active agent forms apart of the fluorogenic moiety, as described herein, in which case, “y”is 0.

According to some embodiments, the polymeric system further comprisesbackbone units A₅ as described herein, and the mol percent is definedaccordingly, as shown herein for Formula IA.

According to some of any of the embodiments described herein, thebackbone units form a polymeric backbone of HPMA co-polymer.

In some of these embodiments, the polymeric system is represented by theFormula 1A as described herein, and further comprises suitable backboneunits comprising the therapeutically active agent, as described herein.

According to some of any of the embodiments described herein, thebackbone units form a polymeric backbone of a PGA polymer.

In some of these embodiments, the polymeric system is represented by theFormula 1B as described herein, and further comprises suitable backboneunits comprising the therapeutically active agent, as described herein.

Processes of Preparing a Polymeric System:

According to an aspect of some embodiments of the present inventionthere is provided a process of preparing a polymeric system whichcomprises a first and a second polymeric moieties, as described herein,the process comprising:

conjugating the fluorogenic compound to a first polymeric backbone inwhich at least a portion of the backbone units have the first cleavablelinking moiety attached thereto and terminate with a first reactivegroup, thereby preparing the first polymeric backbone; and

conjugating the therapeutically active agent to a second polymericbackbone in which at least a portion of the backbone units terminatewith a second reactive group, thereby preparing the second polymericbackbone.

According to some embodiments, the therapeutically active agent isattached to the second polymeric backbone via a cleavable linkingmoiety, the process comprising conjugating the therapeutically activeagent to a second polymeric backbone in which the portion of thebackbone units have the second cleavable linking attached thereto,thereby preparing the second polymeric backbone.

According to some embodiments, conjugating the therapeutically activeagent and/or to the fluorogenic moiety to the backbone units comprisesattaching a spacer to the therapeutically active agent and/or to thefluorogenic moiety, and conjugating the spacer to the backbone units.

According to an aspect of some embodiments of the present inventionthere is provided a process of preparing a polymeric system whichcomprises a single polymeric moiety, the process comprising:

polymerizing a first plurality of monomers, at least one portion of themonomers have the first linking moiety attached to and terminate with afirst reactive group for reacting with the fluorogenic moiety or with afluorogenic moiety conjugated to a spacer, to thereby form the firstpolymeric backbone in which a portion of the backbone units terminatewith the first reactive group;

polymerizing a second plurality of monomers, at least a portion of themonomers terminate with a second reactive group for reacting with thetherapeutically active agent or with the therapeutically active agentconjugated to a spacer, to thereby form the second polymeric backbone inwhich a portion of the backbone units terminate with the second reactivegroup; and

attaching the fluorogenic moiety to the first reactive group and thetherapeutically active agent to the second reactive group,

thereby providing the polymeric system.

The copolymerization of the various monomers can be effected by anypolymerization method known in the art, using suitable polymerizationinitiators and optionally chain transfer agents. Such suitablepolymerization initiators and chain transfer agents can be readilyidentified by a person skilled in the art.

Using the RAFT approach enables to perform the copolymerization at roomtemperature.

The “reversible addition-fragmentation chain transfer” (RAFT)polymerization technique typically involves the use of thiocarbonylthiocompounds, such as dithioesters, dithiocarbamates, trithiocarbonates,and xanthates in order to mediate the polymerization via a reversiblechain-transfer process. This allows access to polymers with lowpolydispersity and high functionality.

In some embodiments, the reactive groups can be protected prior to therespective conjugation thereto. In such cases, the process furthercomprises deprotecting the reactive group prior to the respectiveconjugation.

This allows a regio-controlled conjugation of, for example, theanti-angiogenesis agent to those backbone units that comprises abiodegradable linker.

According to some embodiments, the polymerizing or co-polymerizing isperformed via RAFT polymerization.

Exemplary processes are described in detail in the Examples section thatfollows. These processes can be utilized with any of the fluorogenicmoieties, therapeutically active agents and quenching agents asdescribed herein.

The exemplary processes described in the Examples section that followsand accompanying figures, can be manipulated as desired to suit theselected cleavable linking moiety or moieties, therapeutically activeagent and fluorogenic/fluorescent moiety (and the selected Turn-ONmechanism).

Additional Polymeric Systems:

According to an aspect of some embodiments of the present inventionthere is provided a polymeric system which comprises a fluorogeniccyanine moiety covalently attached via a cleavable linking moiety to aquenching agent, such that upon cleavage of the linking moiety, afluorescent cyanine moiety is generated. In some embodiments, the systemfurther comprising a polymeric moiety attached to the fluorogeniccyanine moiety.

According to some of any of the embodiments described herein, thepolymeric system is represented by a formula selected from Formula VA orVB:

wherein:

Z₁ and Z₂ are each independently a substituted or unsubstitutedheterocylic moiety, as described herein;

R₁ and R₂ are each independently a polymeric moiety;

n is an integer of from 1 to 10;

R′ and R″ are each independently hydrogen, a substituted orunsubstituted alkyl and a substituted or unsubstituted cycloalkyl, or,alternatively, R′ and R″ form together an aryl;

S₅ and S'₅ are each independently a degradable spacer, as describedherein, or absent;

L₅ is the cleavable linking moiety; and

Q is the quenching agent.

The cleavable moiety can be any of the cleavable moieties describedherein.

According to some of any of the embodiments described herein, thecyanine moiety is attached to the polymeric moiety via a spacer,preferably a degradable spacer as described herein.

In some embodiments, the fluorogenic moiety described herein is aFRET-based system, as described herein, preferably a pair-FRET system.

Modular FRET systems previously described in the art (e.g., Redy et al.supra), can be conjugated according to these embodiments, via adegradable spacer, to a polymeric moiety.

In some of these embodiments, the polymeric moiety is PEG. Otherpolymeric moieties, for example, as described herein, are contemplated.

According to some of any of the embodiments described herein, thepolymeric system further comprises a therapeutically active agent,wherein:

(i) the therapeutically active agent is attached to the cleavablelinking moiety, such that upon its cleavage, the therapeutically activeagent is released;

(ii) the therapeutically active agent is attached to the degradablespacer, such that upon its cleavage, the therapeutically active agent isreleased; or

(iii) the therapeutically active agent is attached to a second polymericmoiety, for forming a combined polymeric system, similarly to any of theother embodiments described herein.

Applications:

According to an aspect of some embodiments of the present inventionthere is provided a polymeric system as described in any one of theembodiments described herein, where the system comprises atherapeutically active agent, for use in the treatment and diagnosis ofa medical condition treatable by the therapeutically active agent, orfor use in the preparation of a medicament for treating the medicalcondition.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a medical condition, the methodcomprising administering to a subject in need thereof a polymeric systemas described herein, which comprises a therapeutically active agent thatis usable in treating the medical condition.

According to some of any of the embodiments described herein, themedical condition is cancer.

According to some embodiments of the invention, the therapeuticallyactive agent is an anti-cancer agent.

In some of these embodiments, the therapeutically active agent is ananti-tumor agent (an anti-cancer agent, an anti-proliferative agent, ananti-angiogenesis agent, a chemotherapeutic agent), such as paclitaxel(PTX), as exemplified herein.

According to an aspect of some embodiments of the present inventionthere is provided a polymeric system according to any one of theembodiments described herein, for use in the treatment and diagnosis ofa medical condition treatable by the therapeutically active agent.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating and monitoring a medicalcondition treatable by the therapeutically active agent.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating and monitoring a medicalcondition treatable by the therapeutically active agent, byadministering the polymeric system as described herein to a subject inneed of such treatment.

According to an aspect of some embodiments of the present inventionthere is provided a use of a polymeric system as described herein forpreparing a medicament for treating and monitoring a medical conditiontreatable by the therapeutically active agent.

According to some embodiments of the invention, the medical condition iscancer, the therapeutically active agent is an anti-tumor agent and thecleavable linking moiety/moieties are cleavable by an enzyme expressedor overexpressed in tumor tissues.

According to some embodiments of the present invention, treatment andmonitoring or diagnosis are performed simultaneously, and thus may allowreal-time monitoring and evaluation of the treatment.

The terms “cancer” and “tumor” are used interchangeably herein todescribe a class of diseases in which a group of cells displayuncontrolled growth (division beyond the normal limits). The term“cancer” encompasses malignant and benign tumors as well as diseaseconditions evolving from primary or secondary tumors. The term“malignant tumor” describes a tumor which is not self-limited in itsgrowth, is capable of invading into adjacent tissues, and may be capableof spreading to distant tissues (metastasizing). The term “benign tumor”describes a tumor which is not malignant (i.e. does not grow in anunlimited, aggressive manner, does not invade surrounding tissues, anddoes not metastasize). The term “primary tumor” describes a tumor thatis at the original site where it first arose. The term “secondary tumor”describes a tumor that has spread from its original (primary) site ofgrowth to another site, close to or distant from the primary site.

Non-limiting examples of therapeutically active agents that can beefficiently incorporated in the herein described polymeric systemsinclude amino containing chemotherapeutic agents such as daunorubicin,doxorubicin, N-(5,5-diacetoxypentyl)doxorubicin, anthracycline,mitomycin C, mitomycin A, 9-amino camptothecin, aminopertin,antinomycin, N⁸-acetyl spermidine,1-(2-chloroethyl)-1,2-dimethanesulfonyl hydrazine, bleomycin,tallysomucin, and derivatives thereof; hydroxy containingchemotherapeutic agents such as etoposide, camptothecin, irinotecaan,topotecan, 9-amino camptothecin, paclitaxel, docetaxel, esperamycin,1,8-dihydroxy-bicyclo [7.3.1] trideca-4-ene-2,6-diyne-13-one, anguidine,morpholino-doxorubicin, vincristine and vinblastine, and derivativesthereof, sulfhydril containing chemotherapeutic agents and carboxylcontaining chemotherapeutic agents. Any other anti-cancer agents arealso contemplated.

Other therapeutically active agents that can be beneficiallyincorporated in the herein described polymeric systems include, forexample, antihistamines, anesthetics, analgesics, anti-fungal agents,antibiotics, anti-inflammatory agents, vitamins and anti-infectiousagents.

It is expected that during the life of a patent maturing from thisapplication many relevant cyanine dyes, fluorogenic moieties,therapeutically active agent and/or polymers will be developed and thescope of the terms cyanine dye, cyanine-like structure, polymericbackbone and therapeutically active agent, is intended to include allsuch new technologies a priori.

General:

The term “alkyl” describes a saturated aliphatic hydrocarbon includingstraight chain and branched chain groups. Preferably, the alkyl grouphas 1 to 20 carbon atoms. Whenever a numerical range; e.g., “1-20”, isstated herein, it means that the group, in this case the alkyl group,may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up toand including 20 carbon atoms. In some embodiments, the alkyl group has1-10 carbon atoms. In some embodiments, the alkyl group has 1-4 carbonatoms. Exemplary alkyl groups include, but are not limited to methyl,ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl,undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, heptadecyl,octadecyl and nonadecyl.

The term “cycloalkyl” describes an all-carbon monocyclic or fused ring(i.e., rings which share an adjacent pair of carbon atoms) group whereinone of more of the rings does not have a completely conjugatedpi-electron system. Examples, without limitation, of cycloalkyl groupsare cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane,cyclohexadiene, cycloheptane, cycloheptatriene, and adamantane.

The term “aryl” describes an all-carbon monocyclic or fused-ringpolycyclic (i.e., rings which share adjacent pairs of carbon atoms)groups having a completely conjugated pi-electron system. Examples,without limitation, of aryl groups are phenyl, naphthalenyl andanthracenyl. The aryl group may be substituted or unsubstituted.

The term “heteroaryl” describes a monocyclic or fused ring (i.e., ringswhich share an adjacent pair of atoms) group having in the ring(s) oneor more atoms, such as, for example, nitrogen, oxygen and sulfur and, inaddition, having a completely conjugated pi-electron system. Examples,without limitation, of heteroaryl groups include pyrrole, furane,thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine,quinoline, isoquinoline and purine.

The term “heteroalicyclic” describes a monocyclic or fused ring grouphaving in the ring(s) one or more atoms such as nitrogen, oxygen andsulfur. The rings may also have one or more double bonds. However, therings do not have a completely conjugated pi-electron system.

The term “hydroxy” describes an —OH group.

The term “alkoxy” describes both an —O-alkyl and an —O-cycloalkyl group,as defined herein.

The term “thiol” describes a —SH group.

The term “thioalkoxy” describes both an —S-alkyl group, and an—S-cycloalkyl group, as defined herein.

The term “cyano” describes a —C≡N group.

The term “carbonyl” describes a —C(═O)—R′ group, where R′ is hydrogen,alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) orheteroalicyclic (bonded through a ring carbon) as defined herein.

The term “thiocarbonyl” describes a —C(═S)—R′ group, where R′ is asdefined herein.

The term “O-carbamyl” describes an —OC(═O)—NR′R″ group, where R′ is asdefined herein and R″ is hydrogen, alkyl, cycloalkyl, aryl, heteroaryl(bonded through a ring carbon) or heteroalicyclic (bonded through a ringcarbon) as defined herein.

The term “N-carbamyl” describes an R′OC(═O)—NR″— group, where R′ and R″are as defined herein.

The term “O-thiocarbamyl” describes an —OC(═S)—NR′R″ group, where R′ andR″ are as defined herein.

The term “N-thiocarbamyl” describes an R″OC(═S)NR′— group, where R′ andR″ are as defined herein.

The term “C-amido” describes a —C(═O)—NR′R″ group, where R′ and R″ areas defined herein.

The term “N-amido” describes an R′C(═O)—NR″ group, where R′ and R″ areas defined herein.

The term “C-carboxy” describes a —C(═O)—O—R′ groups, where R′ is asdefined herein.

The term “O-carboxy” describes an R′C(═O)—O— group, where R′ is asdefined herein.

The term “nitro” group describes an —NO₂ group.

The term “amino” group describes an —NH₂ group.

The term “sulfonyl” group describes an —S(═O)₂—R′ group, where R′ is asdefined herein.

The term “halogen” or “halo” describes fluoro, chloro, bromo or iodoatom.

Herein, the phrase “therapeutically active agent” is also referred toherein as “drug”.

The polymeric moieties described herein may possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are encompassed within thescope of the present invention.

As used herein, the term “enantiomer” describes a stereoisomer of acompound that is superposable with respect to its counterpart only by acomplete inversion/reflection (mirror image) of each other. Enantiomersare said to have “handedness” since they refer to each other like theright and left hand. Enantiomers have identical chemical and physicalproperties except when present in an environment which by itself hashandedness, such as all living systems.

The polymeric moieties described herein can exist in unsolvated forms aswell as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention.

The term “solvate” refers to a complex of variable stoichiometry (e.g.,di-, tri-, tetra-, penta-, hexa-, and so on), which is formed by asolute (the conjugate described herein) and a solvent, whereby thesolvent does not interfere with the biological activity of the solute.Suitable solvents include, for example, ethanol, acetic acid and thelike.

The term “hydrate” refers to a solvate, as defined hereinabove, wherethe solvent is water.

As used herein, a “reactive group” describes a chemical group that iscapable of reacting with another group so as to form a chemical bond,typically a covalent bond. Optionally, an ionic or coordinative bond isformed.

A reactive group is termed as such if being chemically compatible with areactive group of an agent or moiety that should be desirably attachedthereto. For example, a carboxylic group is a reactive group suitablefor conjugating an agent or a moiety that terminates with an aminegroup, and vice versa.

A reactive group can be inherently present in the monomeric unitsforming the backbone units, or be generated therewithin by terms ofchemical modifications of the chemical groups thereon or by means ofattaching to these chemical groups a spacer or a linker that terminateswith the desired reactive group.

The term “subject” (alternatively referred to herein as “patient”) asused herein refers to an animal, preferably a mammal, most preferably ahuman, who has been the object of treatment, observation or experiment.

In any of the methods and uses described herein, any of the polymericmoieties described herein can be provided to an individual either perse, or as part of a pharmaceutical composition where it is mixed with apharmaceutically acceptable carrier.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the polymeric moieties described herein (as activeingredient), or physiologically acceptable salts or prodrugs thereof,with other chemical components including but not limited tophysiologically suitable carriers, excipients, lubricants, bufferingagents, antibacterial agents, bulking agents (e.g. mannitol),antioxidants (e.g., ascorbic acid or sodium bisulfite),anti-inflammatory agents, anti-viral agents, chemotherapeutic agents,anti-histamines and the like. The purpose of a pharmaceuticalcomposition is to facilitate administration of a compound to a subject.The term “active ingredient” refers to a compound, which is accountablefor a biological effect.

The terms “physiologically acceptable carrier” and “pharmaceuticallyacceptable carrier” which may be interchangeably used refer to a carrieror a diluent that does not cause significant irritation to an organismand does not abrogate the biological activity and properties of theadministered compound.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of adrug. Examples, without limitation, of excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore pharmaceutically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient's condition(see e.g., Fingl et al., 1975, in “The Pharmacological Basis ofTherapeutics”, Ch. 1 p. 1).

The pharmaceutical composition may be formulated for administration ineither one or more of routes depending on whether local or systemictreatment or administration is of choice, and on the area to be treated.Administration may be done orally, by inhalation, or parenterally, forexample by intravenous drip or intraperitoneal, subcutaneous,intramuscular or intravenous injection, or topically (includingophtalmically, vaginally, rectally, intranasally).

Formulations for topical administration may include but are not limitedto lotions, ointments, gels, creams, suppositories, drops, liquids,sprays and powders. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like may be necessary ordesirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, sachets, pills,caplets, capsules or tablets. Thickeners, diluents, flavorings,dispersing aids, emulsifiers or binders may be desirable.

Formulations for parenteral administration may include, but are notlimited to, sterile solutions which may also contain buffers, diluentsand other suitable additives. Slow release compositions are envisagedfor treatment.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

The pharmaceutical composition may further comprise additionalpharmaceutically active or inactive agents such as, but not limited to,an anti-bacterial agent, an antioxidant, a buffering agent, a bulkingagent, a surfactant, an anti-inflammatory agent, an anti-viral agent, achemotherapeutic agent and an anti-histamine.

According to an embodiment of the present invention, the pharmaceuticalcomposition described hereinabove is packaged in a packaging materialand identified in print, in or on the packaging material, for use in thetreatment and/or monitoring of a disease or disorder or medicalcondition as described herein.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert.

In any of the methods, uses and compositions described herein, thepolymeric systems described herein can be utilized in combination withadditional therapeutically active agents. Such additional agentsinclude, as non-limiting examples, chemotherapeutic agents,anti-angiogensis agents, hormones, growth factors, antibiotics,anti-microbial agents, anti-depressants, immunostimulants.

As used herein the term “about” refers to ±10%

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

MATERIALS AND METHODS

Materials:

HPMA copolymer-Gly-Phe-Leu-Gly-ONp incorporating 10 mol % of themethacryloyl-Gly-Phe-Leu-Gly-p-nitrophenol ester monomer units and HPMAcopolymer-Gly-Phe-Leu-Gly-ethylenediamine (HPMA-GFLG-en) incorporating10 mol % of the methacryloyl-Gly-Phe-Leu-Gly-ethylenediamine wereobtained from Polymer Laboratories (Church Stretton, U.K.). The HPMAcopolymers have a molecular weight of 31,600 Da and a polydispersity of1.66.

PTX was purchased from Petrus Chemicals and Materials 1986 (LTD)(China).

Dulbecco's modified Eagle's medium (DMEM) and PBS, RPMI 1640, fetalbovine serum (FBS), penicillin, streptomycin, nystatin, 1-glutamine,Hepes buffer, sodium pyruvate, and fibronectin were purchased fromBiological Industries Ltd. (Kibbutz Beit Haemek, Israel).

EGM-2 medium was purchased from Cambrex, USA and endothelial cellsgrowth supplement (ECGS) from Zotal (Israel).

All other chemical reagents, including salts and solvents, werepurchased from Sigma-Aldrich (Rehovot, Israel).

All reactions requiring anhydrous conditions were performed under Argonor N₂ atmosphere. Chemicals and solvents were either AR grade orpurified by standard techniques.

Thin layer chromatography (TLC): silica gel plates Merck 60 F₂₅₄:compounds were visualized by irradiation with UV light.

Flash chromatography (FC): silica gel Merck 60 (particle size0.040-0.063 mm), eluent given in parentheses.

High pressure liquid chromatography (HPLC): C18 5u, 250×4.6 mm, eluentgiven in parentheses.

Preparative HPLC: C18 5u, 250×21 mm, eluent given in parentheses.

¹H-NMR spectra were measured using Bruker Avance operated at 400 MHz asmentioned. ¹³C-NMR spectra were measured using Bruker Avance operated at400 MHz as mentioned.

Absorption and fluorescence spectra were recorded on Spectramax-M2fluorescent spectrometer using quartz cuvettes or quartz 96-wells platereader.

Some Abbreviations:

ACN—Acetonitrile, DCM—Dichloromethane, DMAP—4-Dimethylaminopyridine,DMF—N,N′-Dimethylformamide, EtOAc—Ethylacetate, Hex—n-Hexanes,MeOH—Methanol, THF—Tetrahydrofurane, TFA—Trifluoroacetic acid,Et₃N—Triethylamine, EtOH—Ethyl alcohol, NaOAc—Sodium acetate,Ac₂O—Acetic anhydride, DCC—N,N′-Dicyclohexylcarbodiimide, AcOH—Aceticacid.

Dynamic Light Scattering (DLS) Analysis and Surface Charge Measurements:

The mean hydrodynamic diameter of the HPMA copolymer-PTX conjugate andthe zeta-potential measurements were performed using a ZetaSizer Nano ZSinstrument with an integrated 4Mw He—Ne laser (λ=532 nm; MalvernInstruments Ltd., Malvern, Worcestershire, UK). HPMA copolymer-PTXsample were prepared by dissolving 1 mg of polymer conjugate in 1 ml of15.5 mM phosphate buffer, pH=7.4. The polymer solution was vortexed andthen filtered through 0.2 μM filter. All measurements were performed at25° C. using polystyrol/polystyrene (10×4×45) mm cell for DLS analysisand folded capillary cell (DTS 1070) for zeta-potential measurements.

Cathepsin B Activity Assays:

PTX and Cy5 enzymatically-directed release from the conjugates wasstudied in vitro, upon incubation at 37° C. with Cathepsin B (1 unit/ml)in freshly prepared activity phosphate buffer (0.1 M, pH=6.0),containing 0.05 M NaCl, 1 mM Ethylenediaminetetraacetic acid (EDTA) and5 mM reduced glutathione (GSH). As a control, conjugates were incubatedin the absence of Cathepsin B in activity phosphate buffer (0.1 M,pH=6.0), containing 0.05 M NaCl, 1 mM Ethylenediaminetetraacetic acid(EDTA) and 5 mM reduced glutathione (GSH), and/or in Dulbecco's PBS (pH7.4).

Release of Cy5 from HPMA Copolymer-Cy5 Conjugate:

Free Cy5 release was monitored by measuring the change in thefluorescence intensity at sequential time points. The fluorescencemeasurements were carried out at excitation wavelengths of 600 nm usingSpectraMax M5^(e) multi-detection reader. Samples (50 μl) were collectedevery 24 hours (up to 105 hours) and immediately analyzed.

Release of PTX from HPMA Copolymer-PTX-FK Conjugate:

The free PTX release was monitored by reversed phase (RP) HPLC.UltiMate® 3000 Nano LC systems (Dionex) was used, equipped with 3000pump, VWD-3000 UV-Vis detector and Chromeleon® 6.80 software. The columnin use was Phenomenex Jupiter 5μ 250×4.60 mm C-18 300A. Chromatographicconditions were: flow: 1.0 ml/min, gradient: 20% to 100% solution B in20 minutes (sol. A—0.1% TFA in water; sol. B—0.1% TFA in acetonitrile(MeCN)).

Samples (100 μl) were collected every 24 hours, until a plateau wasobserved (up to 50 hours). For PTX extraction, sodium carbonate buffersolution (0.2 M, pH=9.6) was added to each sample, followed by ethylacetate. Samples were vigorously vortex and centrifuged. The organiclayer was carefully removed and evaporated. The residue was dissolved inMeCN and analyzed.

Release of PTX from HPMA Copolymer-PTX Conjugate:

The free PTX release was monitored by reversed phase (RP) HPLC.UltiMate® 3000 Nano LC systems (Dionex) was used, equipped with 3000pump, VWD-3000 UV-Vis detector and Chromeleon® 6.80 software. The columnin use was Phenomenex Jupiter 5μ 250×4.60 mm C-18 300A. Chromatographicconditions were: flow: 1.0 ml/min, gradient: 20% to 100% solution B in20 minutes (sol. A—0.1% TFA in water; sol. B—0.1% TFA in acetonitrile(MeCN)).

Samples (100 μl) were collected every 24 hours, until a plateau wasobserved (up to 50 hours). For PTX extraction, sodium carbonate buffersolution (0.2 M, pH=9.6) was added to each sample, followed by ethylacetate. Samples were vigorously vortex and centrifuged. The organiclayer was carefully removed and evaporated. The residue was dissolved inMeCN and analyzed.

Release of Cy5 from PGA-PTX-Cy5 Conjugate:

Cy5 release was monitored by measuring the change in the fluorescenceintensity at sequential time points. The fluorescence measurements werecarried out at excitation wavelengths of 650 nm using SpectraMax M5^(e)multi-detection reader. Samples (50 μl) were collected every 24 hours(up to 160 hours) and immediately analyzed.

Cell Cultures:

MDA-MB-231 human mammary adenocarcinoma cell line, 4T1 murine mammaryadenocarcinoma cell line and WA239A human melanoma cell line werepurchased from the American Type Culture Collection (ATCC, Manassas,Va., USA).

MDA-MB-231 cells were cultured in DMEM supplemented with 10% FBS, 100μg/mL penicillin, 100 μt/mL streptomycin, 12.5 μl/mL nystatin and 2 mML-glutamine.

4T1 cells were cultured in RPMI 1640 supplemented with 10% FBS, 100mg/mL Penicillin, 100 μl/mL Streptomycin, 12.5 μl/mL Nystatin, and 2 mML-glutamine, 1 mM Sodium pyruvate, 10 mM HEPES buffer and 2.5 g/LD-glucose.

WM239A cells were cultured in RPMI 1640 supplemented with 10% FBS, 100mg/ml Penicillin, 100 μl/ml Streptomycin, 12.5 μl/mg Nystatin, and 2 mML-glutamine.

Cells were grown at 37° C.; 5% CO₂.

Human umbilical vein endothelial cells (HUVEC) were purchased fromLonza, Switzerland and were cultured in EGM-2 medium (Lonza,Switzerland). Cells were grown at 37° C.; 5% CO₂.

For the study of in vitro degradation of HPMA copolymer-Cy5 conjugate,MDA-MB-231 cells (30,000 cells/ml) were seeded to 24-well culture plateswith DMEM supplemented with 10% FBS, 100 μg/mL penicillin, 100 U/mLstreptomycin, 12.5 U/mL nystatin and 2 mM L-glutamine. 24 hours later,HPMA-copolymer-Cy5 (3.8%) at a final concentration of 10 μM eq. Cy5 wasadded. At 0.5, 24 and 48 hours after the addition of the substrate, DMEMwas replaced with PBS and the degradation of the conjugate was monitoredusing SpectraMax M5^(e) multi-detection reader. Non-treated MDA-MB-231cells were used as a control.

Cell Viability Assay:

For the study of HPMA copolymer-PTX conjugate antitumor activity:

4T1 cells (3,000 cells/well), HUVEC (10,000 cells/well) and MDA-MB-231cells (10,000 cells/well) were plated onto 24-well culture plates inRPMI supplemented with 2% FBS, EBM-2 supplemented with 5% FBS or in DMEMsupplemented with 10% FBS respectively, and incubated for 24 hours (37°C.; 5% CO2). The medium was then replaced with RPMI 1640 supplementedwith 10% FBS, EGM-2 or DMEM supplemented with 10% FBS. Cells wereexposed to PTX and PTX bound-conjugates at serial dilutions, atequivalent dose of the free PTX. Number of viable cells was counted by aZ1 Coulter Counter® Cell and Particle Counter (Beckman Coulter®)following 96 hours of incubation.

For the study of PGA-PTX-Cy5 conjugate antitumor activity:

4T1 cells (8,000 cells/wells), WM239A cells (15,000 cells/well) andMDA-MB-231 cells (10,000/well) were plated onto 24-well culture in RPMIsupplemented with 10% FBS, or in DMEM supplemented with 10% FBSrespectively, and incubated for 24 hours (37° C.; 5% CO₂). The mediumwas then replaced with RPMI 1640 supplemented with 10% FBS or DMEMsupplemented with 10% FBS.

Cells were exposed to PTX and PTX-bound conjugates at serial dilutions,at equivalent dose of free PTX. Number of viable cells was counted by aZ1 Coulter Counter® Cell and Particle Counter (Beckman Coulter®)following 72 hours of incubation.

Animals and Tumor Cell Inoculation:

4T1 cells (3×10⁶) were injected subcutaneously (s.c.) into the flank offemale BALB/C mice aged 6-8 weeks). Tumor volume was calculated usingthe standard formula: length×width²×0.52.

Intravital Non-invasive Imaging of Cy5 cathepsin B-dependent release:BALB/c mice bearing subcutaneous 4T1 tumors (about 100 mm³) wereinjected intra-tumorally with HPMA copolymer-Cy5 (0.1 mM; 30 μl) or withequivalent dose of free Cy5. Fluorescent signal within tumor wasassessed at different time points 30 hours following injection usingnon-invasive imaging system CRI Maestro™ Multispectral image-cube wereacquired through 650-800 nm spectral range in 10 nm steps usingexcitation (635 nm longpass) and emission (675 nm longpass) filter set.Mice auto-fluorescence and undesired background signals were eliminatedby spectral analysis and linear unmixing algorithm.

Body Distribution of HPMA Copolymer-SQ-Cy5:

BALB/c mice bearing sub-cutaneous 4T1 tumors (about 300 mm³) wereinjected intravenously (i.v.) with HPMA copolymer-SQ-Cy5 (10 μM; 200μl). Accumulation of the conjugate in the tumor and organs was assessedat different time points for 12 hours. At termination, tumors and organswere excised and imaged. Organs were imaged using non-invasive imagingsystem CRI Maestro™ (filter set—Ex/Em 635/675). Mice auto-fluorescenceand undesired background signals were eliminated by spectral analysisand linear unmixing algorithm. Time dependent tumor contrast profile wasdetermined by the ratio between fluorescence intensities of tumors andthose of normal skin.

Statistical Methods:

Data is expressed as mean±standard deviation (s.d.) for in vitro assaysor ±standard error of the mean (s.e.m.) for in vivo. Statisticalsignificance was determined using an unpaired t-test. All statisticaltests were two-sided. All experiments were performed in triplicates andrepeated at least three times.

Example I

Chemical syntheses of HPMA copolymer conjugates Synthesis of HPMACopolymer-Cy5 conjugate:

The structure of an exemplary HPMA copolymer-Cy5 conjugate is depictedin FIG. 1A. An exemplary synthesis of a HPMA Copolymer-Cy5 conjugate isdepicted in FIG. 2.

Cy5-COOH was synthesized as previously described [Redy, O., et al., Org.Biomol. Chem., 2012. 10(4): p. 710-5].

Cy5-COOH fluorophore was conjugated with HPMA copolymer-GFLG-en intwo-step synthesis, as follows. First, Cy5-COOH (15.1 mg, 0.023 mmol)was dissolved in 0.7 mL anhydrous N,N-Dimethylformamide (DMF).N-Hydroxysuccinimide (NHS) (5.3 mg, 0.046 mmol) andN,N′-dicyclohexylcarbodiimide (DCC) (9.5 mg, 0.046 mmol) were added inorder to activate the free carboxylic group of the fluorophore, forfurther coupling to the HPMA copolymer. The reaction mixture was stirredat room temperature (rT) in dark for 12 hours. Then, HPMA-GFLG-encopolymer (21.1 mg, 0.114 mmol) was dissolved in 0.5 mL anhydrous DMFand added to the reaction mixture. Following the reaction by HighPressure Liquid Chromatography (HPLC) (UltiMate® 3000 Nano LC systems,Dionex), the precipitate was washed with acetone and dried under vacuum.

Purification of the conjugate by size exclusion chromatography (SEC) wasperformed using AKTA/FPLC system (Pharmacia/GE Healthcare), HiTrapDesalting columns (Sephadex G-25 Superfine) in DDW, flow rate 1.0ml/min; UV detection.

In order to remove all excess of free fluorophore, the residue wasdissolved in water and dialyzed for 1 day at 4° C. (MWCO 6-8 kDa)against DI water. The conjugate was isolated by freeze-drying.

Cy5 loading was determined using SpectraMax M5^(e) multi-detectionreader. The absorbance of conjugated Cy5 was measured and compared tothat of free Cy5.

Quenching efficiency was expressed as a percentage of the fluorescenceintensity of the HPMA copolymer-Cy5 conjugate (λ_(Em)=670 nm) comparedwith the emission of the free Cy5 at the equivalent concentration, asshown in Example 5 below and FIG. 11A.

Synthesis of HPMA Copolymer-PTX Conjugate:

The structure of an exemplary HPMA copolymer-PTX conjugate is depictedin FIG. 1B. Paclitaxel (PTX) was conjugated with HPMA copolymer-GFLG-enin two-step synthesis, as depicted in FIG. 3. First, PTX was activatedusing 4-Nitrophenyl chloroformate (PNP-Cl) in order to form PTX-ONp. PTX(107.2 mg, 0.125 mmol) was dissolved in 1 ml pre-distilledTetrahydrofuran (THF) and was stirred at −30° C. Triethylamine (Et₃N)(140 μl, 1.0 mmol) and a grain of 4-Dimethylaminopyridine (DMAP) weredissolved in the dry solvent and added to the reaction mixture. PNP-Cl(151.2 mg, 0.750 mmol) was dissolved in another 1 ml of THF and added tothe reaction. The reaction was followed by Thin Layer Chromatography(TLC) and quenched with 1M HCl at −30° C. The product was extracted fromthe aqueous media using ethyl acetate and purified by silica gel column.

Then, PTX-ONp was conjugated to the HPMA-GFLG-en copolymer in thepresence of Et₃N and Nitrogen atmosphere. The PTX content of the HPMAcopolymer-PTX conjugate was determined by HPLC analysis, at λ=270 nm,against a calibration curve for free PTX.

Synthesis of HPMA Copolymer-PTX-FK Conjugate:

The structure of an exemplary HPMA copolymer-PTX-FK conjugate isdepicted in FIG. 1C. The synthesis of HPMA copolymer-PTX-FK conjugate isillustrated in FIG. 4.

The conjugation of PTX with HPMA copolymer was performed as previouslydescribed [Duncan, R., et al., J Control Release, 2001. 74(1-3): p.135-46]. Briefly,

PTX (168.5 mg, 0.197 mmol) was first attached to an FK-PABC linker(147.5 mg, 0.197 mmol) and the obtained FK-PABC-PTX was conjugated toHPMA copolymer-GFLG-ONp. L-Boc-Phe-ONp was conjugated to L-Lys(alloc)-OH to afford Compound 2. Amidation with 4-aminobenzyl alcohol(PABA) afforded Compound 3, and was followed by activation withp-nitrophenol to afford Compound 4, which was then reacted with PTX toafford Compound 5. Deprotection of the Boc group afforded the freeamine, which was then conjugated with HPMA copolymer-GFLG-ONp, asdepicted in FIG. 4. Finally, deprotection of the alloc group of theamine residue of Lys afforded the desired HPMA copolymer-PTX-FK, thestructure of which is depicted as the final product in FIG. 4 and inFIG. 1C.

The PTX-FK content of the HPMA copolymer-PTX-FK conjugate was determinedby HPLC analysis. The PTX-FK content was determined against acalibration curve for free PTX-FK.

Preparation of Compound 2:

L-Boc-Phe-ONp (104.3 mg, 0.27 mmol) was dissolved in 2 mL DMF. Thencommercially available L-Lys(alloc)-OH (62 mg, 0.27 mmol) and Et₃N (100μL) were added. The reaction mixture was stirred for 12 hours and wasmonitored by TLC (AcOH:MeOH:EtOAc 0.5:10:89.5). Upon completion of thereaction the solvent was removed under reduced pressure and the crudeproduct was purified using column chromatography on silica gel(AcOH:MeOH:EtOAc 0.5:10:89.5) to give compound 2 (107 mg, 83%) as awhite solid (FIG. 4).

Preparation of Compound 3:

Compound 2 (832.1 mg, 1.74 mmol) was dissolved in dry THF and thesolution was cooled to −15° C. Then NMM (0.19 mL, 1.74 mmol) andisobutyl chloroformate (0.27 mL, 2.09 mmol) were added. The reaction wasstirred for 20 minutes and a solution of 4-aminobenzyl alcohol (321.85mg, 2.61 mmol) in dry THF was added. The reaction mixture was stirredfor 2 hours and was monitored by TLC (EtOAc 100%). Upon completion ofthe reaction, the solvent was removed under reduced pressure and thecrude product was purified using column chromatography on silica gel(EtOAc 100%) to give compound 3 (835 mg, 82%) as a yellow solid (FIG.4).

Preparation of Compound 4:

Compound 3 (353.6 mg, 0.60 mmol) was dissolved in dry THF and thesolution was cooled to 0° C. Then DIPEA (0.42 mL, 2.42 mmol),PNP-chloroformate (367 mg, 1.82 mmol) and a catalytic amount of pyridinewere added. The reaction was stirred for 2 hours and monitored by TLC(EtOAc:Hex 3:1). Upon completion of the reaction, the solvent wasremoved under reduced pressure. The crude product was diluted with EtOAcand washed with saturated NH₄Cl. The organic layer was dried overmagnesium sulfate and the solvent was removed under reduced pressure.The crude product was purified using column chromatography on silica gel(EtOAc:Hex 3:1) to give compound 4 (453.2 mg, 79%) as a white solid(FIG. 4).

Preparation of Compound 5:

Compound 4 (360.3 mg, 0.48 mmol) was dissolved in dry DCM. Then PTX(494.06 mg, 0.57 mmol) and DMAP (70.61 mg, 0.57 mmol) were added. Thereaction mixture was stirred for 8 hours and monitored by TLC (EtOAc100%). Upon completion of the reaction, the solvent was removed underreduced pressure and the crude product was purified using columnchromatography on silica gel (EtOAc 100%) to give compound 5 (662 mg,94%) as a white solid (FIG. 4).

Preparation of HPMA Copolymer-PTX-FK (Alloc):

Compound 5 (12 mg, 7.57 μmol) was dissolved in 0.5 mL TFA and stirredfor 2 minutes at 0° C. The excess of acid was removed under reducedpressure and the crude amine salt was dissolved in 0.5 mL DMF. HPMAcopolymer (26.3 mg, ONp=8.32 μmol) was added, followed by the additionof Et₃N (3 μL). The reaction mixture was stirred for 12 hours and thesolvent was removed under reduced pressure.

Free PTX, FK and ONp were removed by FPLC using XK26/70 column withSephadex LH₂O column (MeOH 100%, 1 mL/1 minute) to give the allocprotected HPMA copolymer-PTX-FK as a white solid (20 mg) (FIG. 4).

Preparation of HPMA Copolymer-PTX-FK:

Alloc protected HPMA copolymer-PTX-FK (30 mg, alloc=max. 9.9 μmol) wasdissolved in DMF (1 mL). Then acetic acid (2.71 μL, 47.4 μmol), Bu₃SnH(30.6 μL, 113 μmol) and a catalytic amount of Pd(PPh₃)₄ were added. Thereaction mixture was stirred for 2 hours and was concentrated underreduced pressure, followed by addition of 10 mL of acetone. Theprecipitate was filtered out and washed with acetone several times. Thecrude product was purified by HPLC using XK26/70 column with SephadexLH₂O (MeOH 100%, 1 mL/1 minute) to give HPMA copolymer-PTX-FK (20 mg) asa white solid (FIGS. 1D and 4).

Synthesis of HPMA Copolymer-PTX-Cy5 Conjugate:

The structure of exemplary HPMA copolymer-PTX-Cy5 is depicted in FIG.1D. The synthesis of the HPMA copolymer-PTX-Cy5 conjugate is depicted inFIG. 5.

Cy5-COOH was synthesized as described hereinabove. Next, Cy5-COOHfluorophore was conjugated with HPMA copolymer-GFLG-en in two-stepsynthesis. First, Cy5-COOH (15.1 mg, 0.023 mmol) was dissolved in 0.7 mLanhydrous N,N-Dimethylformamide (DMF). N-Hydroxysuccinimide (NHS) (5.3mg, 0.046 mmol) and N,N′-dicyclohexylcarbodiimide (DCC) (9.5 mg, 0.046mmol) were added in order to activate the free carboxylic group of thefluorophore, for further coupling to the HPMA copolymer. The reactionmixture was stirred at room temperature (RT) in dark for 12 hours. Then,HPMA-GFLG-en copolymer (21.1 mg, 0.114 mmol) was dissolved in 0.5 mLanhydrous DMF and added to the reaction mixture. At reactiontermination, PTX-ONp, prepared as described hereinabove, was added tothe reaction round bottom flask. Following the reaction completion (asmonitored by High Pressure Liquid Chromatography (HPLC) (UltiMate® 3000Nano LC systems, Dionex), the precipitate was washed with acetone anddried under vacuum.

Free Cy5 and PTX-ONp were removed by FPLC using XK26/70 column withSephadex LH20 column (MeOH 100%, 1 mL/1 min); UV detection.

The conjugate was isolated by freeze-drying.

Cy5 loading was determined using SpectraMax M5^(e) multi-detectionreader. The absorbance of conjugated Cy5 was measured and compared tothat of free Cy5.

The PTX content of the HPMA copolymer-PTX-Cy5 conjugate was determinedby HPLC analysis. The PTX content was determined against a calibrationcurve for free PTX.

Quenching efficiency was expressed as a percentage of the fluorescenceintensity of the HPMA copolymer-Cy5 conjugate (λ_(Em)=670 nm) comparedwith the emission of the free Cy5 at the equivalent concentration, as isdetailed hereinunder.

Synthesis of HPMA Copolymer-PTX-FK-Cy5 Conjugate:

The structure of an exemplary HPMA copolymer-PTX-FK-Cy5 is shown in FIG.1E. The synthesis of the HPMA copolymer-PTX-FK-Cy5 is illustrated inFIG. 6.

Previously synthesized L-Boc-Phe-ONp was reacted with L-Lys(alloc)-OH,as described hereinabove, to give dipeptide compound 2. The latter wasconjugated with 4-aminobenzyl alcohol as described hereinabove togenerate alcohol compound 3. Activation of alcohol compound 3 withp-nitrophenyl chloroformate as described hereinabove afforded carbonatecompound 4, which was reacted with PTX as described hereinabove to yieldcompound 5. Deprotection of Boc-Cy5 with TFA, followed by conjugationwith HPMA copolymer-Gly-Phe-Leu-Gly-ONp gave compound 6. Deprotection ofBoc-Phe-Lys(alloc)-PABC-PTX 5 with TFA, followed by conjugation withHPMA copolymer-Cy5 6 gave compound 7. Both Gly-Phe-Leu-Gly and Phe-Lyscathepsin B-cleavable peptides were used in order to provide convenientconjugation chemistry, longer spacer and higher probability of cleavage.Deprotection of the alloc residue of 7 afforded the desired conjugate 1(see, FIG. 1E).

Preparation of Compound 6:

Cy5 (20 mg, 25.41 μmol) was dissolved in 0.5 mL TFA and the solution wasstirred for 5 minutes at 0° C. The excess of acid was removed underreduced pressure and the crude amine salt was dissolved in 0.5 mL DMF.HPMA copolymer-Gly-Phe-Leu-Gly-ONp (24 mg, ONp=12.7 μmol) was addedfollowed by the addition of Et₃N (5 μL). The reaction mixture wasstirred for 12 hours and the solvent was removed under reduced pressure.The crude product was used for the next step without furtherpurification (FIG. 6).

Preparation of Compound 7:

Compound 5 (12 mg, 7.57 μmol) was dissolved in 0.5 mL TFA and thesolution was stirred for 2 minutes at 0° C. The excess of acid wasremoved under reduced pressure and the crude amine salt was dissolved in0.5 mL DMF. Compound 6 (26.3 mg, ONp=8.32 μmol) was added followed bythe addition of Et₃N (3 μL). The reaction mixture was stirred for 12hours and the solvent was removed under reduced pressure.

Free amine (Cy5 and PTX FK), ONp and Cy5 were removed by FPLC usingXK26/70 column with Sephadex LH20 column (MeOH 100%, 1 mL/1 min) to givecompound 7 as a white solid (20 mg) (FIG. 6).

Preparation of Compound I:

Compound 7 (30 mg, alloc=max. 9.9 μmol) was dissolved in DMF (1 mL).Then acetic acid (2.71 μL, 47.4 μmol), Bu₃SnH (30.6 μL, 113 μmol) and acatalytic amount of Pd(PPh₃)₄ were added. The reaction mixture wasstirred for 2 hours and was concentrated under reduced pressure,followed by addition of 10 mL of acetone. The precipitate was filteredout and washed with acetone several times. The crude product waspurified by HPLC using XK26/70 column with Sephadex LH₂O (MeOH 100%, 1mL/1 min) to give compound 1 (20 mg) as a white solid (FIG. 1E).

Example 2 Chemical Synthesis of PGA-PTX-Cy5 Conjugate

The synthesis of PGA-PTX-Cy5 conjugate is depicted in FIGS. 7A-D and8A-B.

PGA was synthesized via the N-carboxyanydride (NCA) polymerization ofglutamic acid, as shown in FIGS. 7A-C. The synthesized PGA was dissolvedin anhydrous N,N-Dimethylformamide (DMF) and mixed withcarbonyldiimidazole (CDI) coupling reagent in order to activate the freepolymer's carboxyl groups. The reaction mixture was stirred at roomtemperature for 4 hours in basic environment. Then PTX was dissolved inanhydrous DMF and added to the reaction mixture to obtain the PGA-PTXconjugate, through formation of an ester bond. The reaction mixture wasstirred overnight at 4° C. The reaction, shown in FIG. 7D, was followedby High Pressure Liquid Chromatography (HPLC) (UltiMate® 3000 Nano LCsystems, Dionex). At the end of the reaction the precipitate was washedwith acetone:chloroform (1:4) solution and dried under vacuum.

The drug loading (x, FIG. 7D) on a polymer was determined using a HighPressure Liquid Chromatography (HPLC) (UltiMate® 3000 Nano LC systems,Dionex).

The obtained PGA-PTX conjugate was dissolved in an anhydrous DMF andmixed again with carbonyldiimidazole (CDI) coupling reagent in order toactivate the unoccupied polymer's carboxyl groups. The reaction mixturewas stirred at room temperature for 4 hours. The solution was removed toa round-bottom flask containing Cy5-NH₂, which was treated withTrifluoroacetic Acid (TFA) to remove a protecting group (Boc) from it(See, FIG. 8A). The reaction was stirred overnight at room temperaturein basic environment. The reaction, shown in FIG. 8B, was followed byHigh Pressure Liquid Chromatography (HPLC) (UltiMate® 3000 Nano LCsystems, Dionex). Upon completion of the reaction the precipitate waswashed with acetone:chloroform (4:1) mixture and dried under vacuum.

In order to remove the excess of free fluorophore, the residue wasdissolved in NaHCO₂ 0.2M buffer and dialyzed for 1 day at 4° C. (MWCO6-8 kDa) against DI water. The final purification of the conjugate bysize exclusion chromatography (SEC) was performed using AKTA/FPLC system(Pharmacia/GE Healthcare), HiTrap Desalting columns (Sephadex G-25Superfine) in DDW, flow rate 1.0 ml/min; UV detection.

Cy5 loading (y in FIG. 8B) was determined using SpectraMax M5^(e)multi-detection reader. The absorbance of conjugated Cy5 was measuredand compared to that of free Cy5. Quenching efficiency was expressed asa percentage of the fluorescence intensity of the PGA-PTX-Cy5 conjugate(λ_(Em)=670 nm) compared with the emission of the free Cy5 at theequivalent concentration.

Preparation of PGA: The starting PGA was synthesized via theN-carboxyanhydride (NCA) polymerization of glutamic acid. First, NCAglutamate was prepared as described in FIG. 7A with a proposedmechanism. H-Glu(OBzl)-OH was used as a starting material, in which theγCOOH is protected with O-benzyl (OBzl). Then, hexylamine (denoted asR₂—NH₂) initiated polymerization of the NCA of γ-benzyl-L-glutamate(FIG. 7B).

PGA was characterized by GPC, zetasizer and ¹H-NMR. Followingdeprotection of the OBzl protecting group in TFA/HBr/AcOH mixture, thecarboxyl group becomes available for coupling to PTX and Cy5.

Preparation of PGA-PTX:

The anti-cancer drug PTX was conjugated to the PGA polymeric backbonevia its carboxyl groups, as depicted in FIG. 7D. First, PGA functionalgroups were activated by carbonyldiimidazole (CDI) coupling in anhydrousDMF, and then PTX was added to an activated reaction mixture to obtainthe PGA-PTX conjugate, through a formation of an ester bond.

As PGA is a substrate of a lysosomal enzyme cathepsin B, uponendocytosis of the conjugate to the cells it should be cleaved, andresult in release of free PTX in the tumor interstitium.

Preparation of PGA-PTX-Cy5:

Cy5, in a sufficient amount, was conjugated to the polymeric backbone ofPGA, to achieve a fluorophore self-quenching. The Cy5 is attached to thePGA via an amide bond. The polymeric backbone cleavage by the cathepsinB enzyme should release the fluorophore from the conjugate, thusremoving the self-quenching and activating the fluorescence.

PGA-PTX-Cy5 conjugate was synthesized as described in FIGS. 8A-B. First,the unoccupied carboxylic groups of PGA were activated with CDI couplingagent, supported by DMAP as a catalyst in a basic environment and then aCy5-NH₂, after the protecting group (Boc) removal (FIG. 8A), was mixedwith the activated PGA-PTX polymeric conjugate, to form an amide bondand obtain the desired conjugate (FIG. 8B).

Example 3 Characterization and Activity of HPMA Copolymer-PTX Conjugate

Cathepsin B-Mediated Degradation and Release:

Cathepsin B-mediated degradation and release of PTX from HPMAcopolymer-PTX over time is described in FIG. 9B. PTX concentration wasincreased as a function of time and expressed by area under the curve(AUC), and represents a satisfactory efficiency of cathepsin B activity.Release kinetics of PTX release show a complete release from HPMAcopolymer after approximately 80 hours.

Dynamic Light Scattering and Zeta Potential of HPMA Copolymer-PTXConjugate:

The hydrodynamic diameter size distribution and zeta-potential of HPMAcopolymer-PTX conjugate was determined using a ZetaSizer analyzer. Table1 presents physico-chemical characterization of HPMA copolymer-PTXconjugate.

TABLE 1 Total PTX Size Zeta Loading Conjugate Mw (kDa)^(a) (nm)^(b)Potential (mV)^(b) (mol %)^(c) HPMA- 37.70 11.99 3.69 4.0 PTX^(a)theoretical value, ^(b)determined by Zetasizer in 10% PBS (1 mg/mL),^(c)determined by analytical HPLC at λ = 270 nm.

The mean hydrodynamic diameter was 11.99 nm and the zeta potential valuewas 3.69 mV (Table 1). As expected, HPMA copolymer-PTX has a neutralcharge and its size is in the nano range, which enables its targeting tothe tumor via the EPR effect.

HPMA Copolymer-PTX Conjugate Inhibits the Proliferation of 4T1 MammaryAdenocarcinoma and MDA-MB-231 Cancer Cell Lines:

The mitotic inhibitor PTX is a potent cytotoxic agent approved as firstline therapy for breast cancer. The cytotoxic effect of the conjugatewas evaluated on murine 4T1 mammary adenocarcinoma cells. The obtaineddata are presented in FIG. 10A. As shown therein, the proliferation of4T1 cells was inhibited by HPMA copolymer-PTX conjugate with an IC50 ofabout 15 μM (see, FIGS. 10A and 10D).

HPMA copolymer alone served as control and was inert at all theconcentrations tested (data not shown), in agreement with previouslypublished data [Duncan et al., 2001, supra]. IC50 for free PTX was about35 nM (see, FIG. 10D). The difference in IC₅₀ between the free drug andthe conjugate can be attributed to the slow release kinetics of the drugfrom the carrier.

These results are in accordance with previous reports showing thecytotoxic effect of an analogous HPMA copolymer-PTX conjugate on breastcancer cells [Miller, K., et al., Angew Chem Int Ed Engl, 2009. 48(16):p. 2949-54; Miller, K., et al., Mol Pharm, 2011. 8(4): p. 1052-62].

HPMA Copolymer-PTX Exhibits Anti-Angiogenic Effect In Vitro:

The anti-angiogenic effect of PTX on endothelial cells was previouslydemonstrated [Miller et al., 2009 and 2011, supra; Clementi, C., et al.,Mol Pharm, 2011. 8(4): p. 1063-72]. These studies have demonstratedinhibitory effect of PTX on different stages of the angiogeniccascade-proliferation, migration and formation of tube-like structures.Since endothelial cells that construct the tumor vasculature, alsooverexpress cathepsin B, the inhibitory effect of the herein describedcathepsin B-dependent delivery system on HUVEC proliferation wasevaluated.

The obtained results are presented in FIGS. 10B and 10D, and indeed showthat the HPMA copolymer-PTX conjugate exhibited cytotoxic effect onHUVEC proliferation with an IC₅₀ of about 90 nM compared to about 2 nMof the free drug. Similarly to the aforementioned experiments performedon 4T1 cells, HPMA copolymer alone served as control and was nontoxic atall the concentrations tested (data not shown).

The results demonstrate that PTX maintained its cytotoxic activity invitro upon conjugation to HPMA copolymer and that a cathepsinB-dependent release mechanism is efficient for active targeting of HPMAcopolymer-PTX to breast cancer and its vasculature overexpressing theenzyme in vivo.

Example 4 Characterization and Activity of HPMA Copolymer-PTX-FKConjugate

The chemical structure of HPMA copolymer-PTX-FK conjugate is presentedin FIG. 1D. PTX was conjugated to HPMA copolymer through aGly-Phe-Leu-Gly (GFLG) linker and an addition of Phe-Lys-PABC linkerrespectively, both cleavable by cathepsin B enzyme.

The conjugation to the HPMA copolymer was a two-step procedure in whichPTX was first attached to the FK-PABC linker and then conjugated to HPMAcopolymer-GFLG-ONp (FIG. 4).

The resulting conjugate was water-soluble, PTX-FK loading was 1.12 mol %(2.02 PTX molecules per polymeric chain).

Cathepsin B-mediated degradation and release of PTX from HPMAcopolymer-PTX-FK over time is described in FIG. 9A. PTX concentrationwas increased as a function of time and expressed by area under thecurve (AUC), and presents a satisfactory efficiency of cathepsin Bactivity. Release kinetics of PTX show a complete release from HPMAcopolymer after approximately 50 hours.

HPMA Copolymer-PTX-FK Conjugate Inhibits the Proliferation of MDA-MB-231Human Mammary Adenocarcinoma Cancer Cell Line:

The cytotoxic effect of the conjugate was evaluated on human MDA-MB-231mammary adenocarcinoma cells. The obtained data is presented in FIGS.10C and 10D. As shown therein, the proliferation of MDA-MB-231 cells wasinhibited by HPMA copolymer-PTX-FK conjugate with an IC50 of 100 nM.HPMA copolymer alone served as control and was inert at all theconcentrations tested (data not shown), in agreement with previouslypublished data.

IC₅₀ for free PTX was about 0.5 nM. The difference in IC₅₀ between thefree drug and the conjugate can be attributed to the slow releasekinetics of the drug from the carrier. These results are in accordancewith previous reports showing the cytotoxic effect of an analogous HPMAcopolymer-PTX conjugate on breast cancer cells [Miller et al. supra].

Example 5 Characterization and Activity of HPMA Copolymer-SQ-Cy5Conjugate

The chemical structure of the HPMA-copolymer-GFLG-en-Cy5 conjugate, alsoreferred to herein as HPMA copolymer-SQ-Cy5 conjugate(SQ=self-quenching), or simply as HPMA copolymer-Cy5, is presented inFIG. 1A.

HPMA-copolymer-GFLG-en-Cy5 conjugate was synthesized with 3.8 mol %loading (7.5 dye molecules per polymeric chain) and its fluorescencespectrum was characterized, in order to evaluate both the self-quenchingof the conjugated fluorophore and its biodegradability by cathepsin B.

As shown in FIG. 11A, HPMA copolymer-SQ-Cy5 conjugate exhibitssignificant self-quenching; the fluorescent signal of the HPMAcopolymer-SQ-Cy5 conjugate was reduced compared to an equivalentconcentration of free Cy5. At the signal's linear range, the two lineartrend lines slopes of free Cy5 and HPMA copolymer-Cy5 were compared anda reduction of 54% therebetween was obtained. In addition, after thesignal reached saturation, a reduction of about 80% was observed.

In order to evaluate the increase in fluorescence intensity due toenzymatic cleavage, HPMA copolymer-SQ-Cy5 was incubated in the presenceof cathepsin B and release of Cy5 was assessed by fluorescence signal.The results are presented in FIG. 11B, and show that the measuredfluorescence intensity was dramatically increased over time andplateaued after about 100 hours. In the absence of the enzyme, there wasno increase in fluorescent signal.

A HPMA copolymer-SQ-Cy5, with loading of 3.8 mol % Cy5, exhibitedsatisfactory self-quenching properties and activation by cathepsin B.Under physiological conditions, the conjugate is relatively opticallysilent in its quenched state (i.e., turn-OFF), and becomes highlyfluorescent after enzymatic cleavage of the GFLG linker by cathepsin B.It is postulated that the loading of the fluorophore affects the optimalperformance of the conjugate. At low fluorophore loading, only limitedquenching may occur, whereby at high fluorophore loading, the Turn-ONmay not occur as the enzyme may not reach its target site [Melancon, M.P., et al., Pharm Res, 2007. 24(6): p. 1217-24].

In Vitro Turn-on Capacity on HPMA-SQ-Cy5 Conjugate:

As shown in FIG. 11C, incubation of HPMA copolymer-GFLG-Cy5 in culturedMDA-MB-231 cells resulted in significantly higher fluorescence signalintensity than that observed in culture non-treated MDA-MB-231 cellsduring a period of 0.5-48 hours.

In Vivo Characterization of HPMA Copolymer-SQ-Cy5 Cathepsin-DependentRelease:

As mentioned above, overproduction of cathepsin B in vivo is associatedwith breast carcinoma, both tumor cell population and tumor endothelium.Thus, the ability of the probe conjugate to exhibit Turn-ON properties,and to image endogenously produced cathepsin B in a murine model of 4T1breast adenocarcinoma tumors was evaluated.

Mice bearing approximately 100 mm³ tumors were injected intra-tumorallywith 0.1 mM free Cy5 and equivalent Cy5 dose of HPMA copolymer-SQ-Cy5.Injected mice were imaged using CRI Maestro™ non-invasive fluorescenceimaging systems over time for approximately 8 hours.

The obtained data is presented in FIGS. 12A and 12B. The initialfluorescent signal of HPMA copolymer-SQ-Cy5 is significantly lower thanthat of free Cy5, which exhibits the self-quenching properties ofhigh-loaded Cy5. In addition, FIG. 12A clearly shows an increase of1.8-fold change in fluorescence signal within 1 hour of injection.Interestingly, HPMA copolymer-SQ-Cy5 exhibited improved biocompatibilityfor in vivo florescence imaging. While the free Cy5 bleached almostcompletely about 3 hours following injection, although lower, thefluorescent signal of the conjugated Cy5 retained for long period oftime (FIG. 12A). Consequently, HPMA copolymer-SQ-Cy5 may represent asuitable approach for in vivo imaging of endogenous cathepsin B intumor, to indicate on drug release in real time and for tumor monitoringover time.

HPMA Copolymer-SQ-Cy5 Exhibit Improved Pharmacokinetics Profile in Mice:

To assess whether HPMA copolymer-PTX exhibits preferable accumulationand release at the tumor site once injected systemically, HPMAcopolymer-SQ-Cy5 was administered into mice and its pharmacokineticsprofile was utilized to deduce on HPMA copolymer-PTX pharmacokineticsprofile.

Mice bearing about 300 mm³ 4 T1 tumors were administered via the tailvein with HPMA copolymer-Cy5 (10 μM; 200 μl). It was hypothesized thatconjugation will result in half-life prolongation and tumor specificaccumulation and release.

As shown in FIG. 13A, and in accordance with this hypothesis, HPMAcopolymer-SQ-Cy5 demonstrated accumulation in the tumor. Mice wereimaged over time and fluorescent signal in the tumor was measured. Asdescribed in FIG. 13B, at the first 3 hours following administration,HPMA copolymer-Cy5 exhibited no preferable accumulation at the tumor.However, after 4 hours increased fluorescent signal in the tumor wasmeasured.

Next, healthy organs (heart, lungs, liver, spleen and kidneys) andtumors were resected from mice injected with the conjugate at differenttime points and the fluorescent intensity was evaluated. The obtaineddata is presented in FIG. 13C and show that increased fluorescent signalwas measured within tumors 12 hours following administration. HPMAcopolymer-SQ-Cy5 was hardly detectable in the heart and spleen.Interestingly, increased fluorescent signal was also measured in theliver and kidneys. However, since the fluorescent signal in these organsdid not increase over time, it can be concluded that Cy5 was notreleased from HPMA copolymer, hence, PTX will not be released andfluorescent as well. After several hours, the signal decreased in theseorgans and it was only increased over time within tumors. To conclude,HPMA copolymer-SQ-Cy5 exhibited preferable accumulation in the tumor,liver and kidneys, but Cy5 was released, presumably by enzymaticcleavage, only within tumor cells expressing cathepsin B.

Example 6 Characterization and Activity of HPMA Copolymer-PTX-Cy5 andHPMA Copolymer-PTX-FK-Cy5 Conjugates

In HPMA copolymer-PTX-Cy5 conjugate, also referred to herein as HPMAcopolymer-SQ-Cy5-PTX, both Cy5 and PTX were conjugated to HPMA copolymerthrough a Gly-Phe-Leu-Gly (GFLG) linker, cleavable by cathepsin Benzyme. For HPMA copolymer-PTX-FK-Cy5, also referred to herein as HPMAcopolymer-SQ-Cy5-PTX-FK, both Cy5 and PTX were conjugated to HPMAcopolymer through a Gly-Phe-Leu-Gly (GFLG) linker and an addition ofPhe-Lys-PABC linker, respectively, cleavable by cathepsin B enzyme.Characterization of HPMA copolymer-SQ-Cy5-PTX and HPMAcopolymer-SQ-Cy5-PTX-FK conjugates show an increase in fluorescencefollowing incubation with cathepsin B, as presented in FIGS. 14A-B. FIG.14C presents comparative plots showing that fluorescence intensity(λ_(Ex)=650 nm) decreased with increasing load of Cy5.

Example 7 Characterization and Activity of PGA-PTX-Cy5 Conjugate

Characterization:

To characterize the conjugate, its absorption and fluorescence wereevaluated. Cy5 loading on the conjugate was calculated by spectroscopyanalysis.

In addition, these conjugate are substrate for the enzyme cathepsin B.When mixing the conjugate with the enzyme (in a suitable buffer with alow pH), the enzyme should cleave the conjugate and release the attachedmoieties. The enzymatic reaction was followed using HPLC andspectrophotometer. The absorption spectrum of the PGA-PTX-Cy5 conjugaterelative to the absorption spectrum of the free Cy5 is presented in FIG.15A. As shown in the emission spectrum (FIG. 15B), a fluorescent signalobserved for the conjugates of 4 mol % and 7.5 mol % Cy5 loading issignificantly lower relative to a signal emitted from an unconjugatedCy5. The fluorescence intensity decreases as Cy5 loading on a polymerincreases.

Cy5 release from the conjugate was also monitored by measuring thechange in the fluorescence intensity at sequential time points. Thefluorescence measurements were carried out at excitation wavelengths of650 nm using SpectraMax M5^(e) multi-detection reader. Samples (50 μl)were collected every 24 hours (up to 160 hours) and immediatelyanalyzed. The incubation of the PGA-PTX-Cy5 conjugate with cathepsin Benzyme, showed an increase in emitted fluorescent signal as a functionof time. In the absence of cathepsin B, almost no increase influorescence was observed (FIG. 15C). This data shows a self-quenchingability of PGA-PTX-Cy5 conjugate, since while the conjugate is intactthe fluorescent signal is significantly silent, while when the Cy5 isreleased from the PGA polymeric backbone in the presence of cathepsin B,there is no longer self-quenching effect and the fluorescent signalincreases.

Release of PTX from the conjugate incubated with cathepsin B enzyme overtime was monitored by reversed phase (RP) HPLC. UltiMate® 3000 Nano LCsystems (Dionex), equipped with 3000 pump, VWD-3000 UV-Vis detector andChromeleon® 6.80 software. The column in use was Phenomenex Jupiter 5μ250×4.60 mm C-18 300A. Chromatographic conditions were: flow: 1.0ml/min, gradient: 20% to 100% solution B in 20 minutes (sol. A—0.1% TFAin water; sol. B—0.1% TFA in acetonitrile (MeCN)). Samples (50 μl) werecollected simultaneously with samples for Cy5 release determination,every 24 hours (up to 160 hours). To each sample 150 μl of methanoladded and immediately analyzed. The AUC of a PTX peak was increase overthe time as shown in FIG. 15D.

Anti-Proliferative Activity:

The cytotoxic effect of the conjugate was evaluated on human MDA-MB-231mammary adenocarcinoma cells, on murine 4T1 adenocarcinoma cells and onhuman WM239A melanoma cells. As shown in FIGS. 16A-D, the proliferationof all cell lines was inhibited by PGA-PTX-Cy5 conjugate with an IC₅₀ ofabout 40 nM for MDA-MB-231 cells, an IC₅₀ of about 650 nM for 4T1 cellsand with IC₅₀ of about 80 nM for WM239A cells.

Free Paclitaxel and PGA-PTX polymeric conjugate served as controls. IC₅₀for free PTX was about 2 nM in case of MDA-MB-231 cells, about 60 nM incase of 4T1 cells and about 5 nM in case of WM239A cells (FIG. 16D). Thedifference in IC50 between the free drug and the conjugate can beattributed to the slow release kinetics of the drug from the carrier.These results are in accordance with previous reports showing thecytotoxic effect of an analogous PGA-PTX conjugate on breast cancercells.

PGA-PTX-Cy5 conjugate inhibits the migration of HUVEC: The migration ofHUVEC in the presence of PGA-PTX-Cy5 conjugate was evaluated using thescratch assay. The method is based on the observation that, uponcreation of a new artificial gap, so called “scratch”, on a confluentcell monolayer, the cells on the edge of the newly created gap will movetoward the opening to close the “scratch” until new cell-cell contactsare established again. Following 24 hours incubation of HUVEC in 6 wellsplate (500,000 cells per well) the cells were treated with the conjugateand the different controls (such as: PTX, PGA-PTX and no treatment). Attime zero (t=0) images were taken by phase-contrast microscope in areference point. Following another 12 h of incubation, images of thereference point were taken again. The samples were analyzedquantitatively by ImageJ software. The PGA-PTX-Cy5 conjugate, atPTX-equivalent concentrations of 20 nM inhibited efficiently themigration of HUVEC by 36% of gap closure (FIG. 17). As expected, PTXalone, which is known to be anti-angiogenic at low doses, showed higherinhibitory effect of 20% of gap closure.

The effect of PGA-PTX-Cy5 conjugate on the ability of HUVECs to formcapillary-like tube structures on matrigel was also evaluated. Aspreviously reported, such an assay can emulate the capability ofendothelial cells to form vascular networks in vivo. HUVEC wereincubated in the presence of PGA-PTX-Cy5 conjugate, free PTX, free Cy5,and PGA, and in absence of treatment, for 8 hours, pictured andquantitatively analyzed. The obtained data is presented in FIGS. 18A-B.As shown therein, PGA-PTX-Cy5 at PTX load equivalent of 20 nM inhibitedthe tubular structures formation by about 40%, compared to untreatedcells used as negative control.

Example 8 Syntheses of HPMA Copolymer Conjugates by RAFT Polymerization

Reversible addition-fragmentation chain transfer (RAFT) polymerizationis a versatile controlled/“living” free radical polymerization techniqueresulting in predetermined molecular weight with narrow polydispersity.This technique enables the theoretical calculation of molecular weightof the polymers by the ratio of monomer concentration to chain transferagent concentration and the conversion of the polymerization. Additionaladvantage is the ease of manufacturing since the synthesis is carriedout in a one-pot reaction.

Functional monomers were therefore designed and synthesized for RAFTpolymerization of HPMA copolymer conjugates for theranostics. Thisdesign of RAFT synthesized copolymer conjugates benefits from controlledpolymerization and a lower polydispersity that may improve itsbiodistribution and accumulation at the tumor site, and further, thehigher amount of the activatable diagnostic moiety can lead to anincreased signal emitted upon the probe activation to a Turn-ON state.

Preparation of Functional Monomers:

The syntheses of N—(N-Boc-ethylenediamine) methacryloylglycylglycylamide(MA-Gly-Gly-diamine-Boc) andmethacryloylglycylphenylalanylleucylglycyl-p-aminophenylcarbonatep-nitrophenyl ester (MA-Gly-Phe-Leu-Gly-PABC-ONp; MA-GFLG-PABA;MA-Gly-Phe-Leu-Gly-PABA) are shown in FIGS. 19A and 19B, respectively.

Preparation of MA-Gly-Phe-Leu-Gly-OH:

MA-Gly-Phe-Leu-Gly-OH was synthesized by solid phase peptide synthesis(SPPS) and manual Fmoc/tBu strategy using 2 grams of 2-chlorotritylchloride beads with 80% of loading leading to a yield of 0.88 grams,95%.

Preparation of MA-Gly-Phe-Leu-Gly PABA:

MA-GFLG-OH (400 mg, 0.815 mmol) was dissolved in dry THF and thesolution was cooled to −15° C. Then NMM (90 μL, 0.815 mmol) and isobutylchloroformate (128 μL, 0.978 mmol) were added. The reaction was stirredfor 20 minutes and a solution of 4-aminobenzyl alcohol (151 mg, 1.22mmol) in dry THF was added. The reaction mixture was stirred for 12hours and was monitored by TLC (EtOAc 100%). Upon completion of thereaction, the solvent was removed under reduced pressure and the crudeproduct was purified by using column chromatography on silica gel (1-8%MeOH in EtOAc) to give MA-Gly-Phe-Leu-Gly (262 mg, 53%) as a yellowsolid. See, FIG. 19B.

Preparation of MA-Gly-Gly-diamine-Boc:

MA-Gly-Gly-OH was synthesized as described in Rejmanova et al. (1977)Makromol Chem 178, 2159-2168, followed by amination that was carried outas follows: MA-Gly-Gly-OH (200 mg, 0.864 mmol), DCC (196.2 mg, 0.951mmol), NHS (99.5, 10 mmol) were stirred in DMF for 1 hour, thenN-(tert-butoxycarbonyl) (Boc)-ethylenediamine (138.5 mg, 0.864 mmol) wasadded, and the reaction mixture was stirred for 24 hours at roomtemperature. The product was obtained by precipitation in ethyl ether.See, FIG. 19A.

RAFT Polymerization:

Exemplary synthetic schemes utilizing the above-described functionalmonomers are presented in FIGS. 20 and 21. A RAFT synthesized HPMAcopolymer precursor is obtained, bearing functional groups (ONp and/orNH₂-Boc) for facile conjugation of a drug (e.g., paclitaxel) and afluorescent agent (e.g., Cy5 or FITC).

FIG. 20 presents the chemical structure, two-step synthesis and cleavagemechanism by Cathepsin B of HPMA copolymer-Gly-Phe-Leu-Gly-PABC-Cy5-PTX,an exemplary self-quenching (homo-FRET) based theranostic systemsynthesized by RAFT polymerization.

FIG. 21 presents the chemical structure, two-step synthesis and cleavagemechanism by Cathepsin B of HPMAcopolymer-Gly-Phe-Leu-Gly-PABC-FITC-PTX, an exemplary self-quenching(homo FRET) based theranostic system synthesized by RAFT polymerization.

FIG. 22A presents exemplary synthetic schemes for the preparation ofBoc-NH-LG-PABC-PTX, Boc-NH-LG-PABC-Cy5 and Boc-NH-LG-PABC-FITC, usefulfor conjugating PTX, Cy5 and FITC, respectively, as non-limitingexamples of a drug and dyes, to the HPMA copolymer precursor prepared byRAFT polymerization as described hereinabove.

FIG. 22A presents an exemplary synthetic scheme of drug and dyedipeptide-PABC moieties (ivDde-NH—FK-PABC-PTX and ivDde-NH—FK-PABC-Cy5respectively) useful for conjugation to HPMA copolymer-dipeptide-ONp(Gly-Gly-ONp).

The Cy5 is presented herein as a representative example and can bereplaced with other fluorogenic or fluorescent dye moieties or probessuch as QCy7 and FRET probes as described herein.

The PTX can also be replaced by other therapeutically active agents asdescribed herein.

Example 9 FRET-Based Polymeric Systems

Förster Resonance Energy transfer (FRET)-based probes are typicallycomposed of a fluorescent dye attached through a cleavable linker to aquencher, as depicted in FIG. 23. Under such circumstances, the excitedfluorophore transfers its excitation energy to the nearbyquencher-chromophore in a non-radiative manner through long rangedipole-dipole interactions. Cleavage of the linker moiety (e.g., by ananalyte or enzyme of interest), results in diffusion of the fluorescentdye away from the quencher and thereby in generation of a measurablefluorescent signal.

In some exemplary polymeric FRET-based systems according to the presentembodiments, a fluorescent dye is attached to the polymeric backboneunits through a cleavable linker. In such cases, upon cleavage of thelinker, the fluorescent dye molecules diffuse away from one another,thus generating a measurable fluorescent signal. These systems arereferred to herein as self-quenching (SQ) or homo-FRET based systems.When a therapeutically active agent (drug) is also attached to thepolymeric backbone, the system is theranostic. Exemplary such systemsare described hereinabove and are shown in FIGS. 1A-E, 2, 5, 6, 8B, 20and 21.

In other exemplary polymeric FRET-based systems, the fluorescent dye isattached to the polymeric backbone units via a cleavable linker, and aquencher is also attached to the polymeric backbone. The quencher can beattached to some of the polymeric backbone units via a linker(preferably a non-cleavable linker). In some embodiments, thefluorescent dye is attached to a portion of the backbone units and thequencher is attached to another portion of backbone units of thepolymeric backbone (referred to herein also as FRET mode I). In someembodiments, the quencher is attached to the end of the polymericbackbone (referred to herein also as FRET mode II). Exemplary suchsystems are described hereinafter and in FIGS. 24-30.

In other exemplary systems, a moiety composed of a fluorescent dye (as afluorogenic moiety) and a quencher, linked to one another via a linker,is utilized. This moiety can be attached to polymeric backbone units,preferably via a cleavable linker, whereby the system is designed suchthat upon cleavage of the linker, the fluorescent dye diffuses away fromthe quencher and a measurable fluorescent signal is generated (referredto herein as FRET mode III). Exemplary such moieties are describedhereinafter and are shown in FIGS. 34 and 35, and an exemplary polymericsystem comprising such a moiety is shown in FIG. 39. Alternatively, sucha moiety can be attached to the end of the polymeric backbone (referredto herein as FRET mode IV). A polymeric system comprising such a moietyis shown in FIGS. 31A-B.

The present inventors have designed exemplary FRET-based polymericsystems and moieties to be incorporated in such systems as follows.

FRET-Based Systems with HPMA Copolymer Conjugates (FRET Modes I and II):

HPMA copolymer conjugates having a drug (e.g., PTX) and a dye (e.g., afluorogenic moiety such as Cy5 or FITC) attached to the HPMA backboneunits, and further comprising a quencher attached to the polymericbackbone are prepared by RAFT polymerization as described in Example 8hereinabove, and the quencher is attached to the backbone by one of thefollowing approaches: (i) through coupling chemistry of quencher-COOH toa linker of Gly-Gly-NH₂ (see, FIGS. 24, 25, 28 and 29; FRET mode I); or(ii) by coupling chemistry of quencher-NH₂ to a COOH end-functionalizedHPMA-copolymer chain, which results from the rational design of RAFTagent with functional carboxylic acid end group (see, FIGS. 26 and 27;FRET mode II).

FIG. 24 presents the chemical structure, two-step synthesis and cleavagemechanism by cathepsin B of HPMAcopolymer-Gly-Phe-Leu-Gly-PABC-Cy5-Quencher-PTX as an illustration for aFRET-based theranostic system synthesized by RAFT polymerization (FRETmode I). The functional monomers and preparation thereof, forsynthesizing HPMA copolymer precursor are described in Example 8hereinabove and in FIGS. 19A and 19B. The functionalized PTX and Cy5moieties and the preparation thereof are presented in FIGS. 22A-B.

FIG. 25 presents the chemical structure, two-step synthesis and cleavagemechanism by cathepsin B of HPMAcopolymer-Gly-Phe-Leu-Gly-PABC-FITC-DR1-PTX as an illustration for aFRET-based theranostic system synthesized by RAFT polymerization (FRETmode I). The functional monomers and preparation thereof, forsynthesizing HPMA copolymer precursor are described in Example 8hereinabove and in FIGS. 19A and 19B. The functionalized PTX and FITCmoieties and the preparation thereof are presented in FIGS. 22A-B.

FIG. 26 presents the chemical structure, two-step synthesis and cleavagemechanism by Cathepsin B of HPMAcopolymer-Gly-Phe-Leu-Gly-PABC-Cy5-Quencher-PTX as an illustration for aFRET-based theranostic system synthesized by RAFT polymerization. Thequencher-amine is coupled to the COOH end-functionalized HPMAcopolymer-PTX-Cy5 conjugate, providing one quencher molecule perpolymeric chain (FRET mode II). The functional monomers and preparationthereof, for synthesizing HPMA copolymer precursor are described inExample 8 hereinabove and in FIGS. 19A and 19B. The functionalized PTXand Cy5 moieties and the preparation thereof are presented in FIGS.22A-B.

FIG. 27 presents the chemical structure, two-step synthesis and cleavagemechanism by Cathepsin B of HPMAcopolymer-Gly-Phe-Leu-Gly-PABC-FITC-DR1-PTX as an illustration for aFRET-based theranostic system synthesized by RAFT polymerization. Thequencher, DR1-amine is coupled to the COOH end-functionalized HPMAcopolymer-PTX-FITC conjugate, providing one quencher molecule perpolymeric chain (FRET mode II). The functional monomers and preparationthereof, for synthesizing HPMA copolymer precursor are described inExample 8 hereinabove and in FIGS. 19A and 19B. The functionalized PTXand FITC moieties and the preparation thereof are presented in FIGS.22A-B.

FIG. 28 is a scheme depicting a synthesis of HPMAcopolymer-Gly-Phe-Leu-Gly-PABC-Cy5-Quencher-PTX conjugate (FRET mode I),effected by conjugating Boc-NH-LG-PABC-PTX, Boc-NH-LG-PABC-Cy5 and aquencher to HPMA copolymer precursor bearing Gly-Phe-ONp/Gly-Gly-diamineBoc linkers, and addition of LG-PABC linker to PTX and Cy5 followed bytheir addition to the resulting conjugate HPMAcopolymer-Gly-Phe-Leu-Gly-PABC-Cy5-Quencher-PTX. The functional monomersand preparation thereof, for synthesizing HPMA copolymer precursor aredescribed in Example 8 hereinabove and in FIGS. 19A and 19B. Thefunctionalized PTX and Cy5 moieties and the preparation thereof arepresented in FIGS. 22A-B.

FIG. 29 is a scheme depicting a synthesis of HPMAcopolymer-Gly-Phe-Leu-Gly-PABC-FITC-DR1-PTX conjugate (FRET mode I),effected by conjugating Boc-NH-LG-PABC-PTX, Boc-NH-LG-PABC-FITC and DR1to HPMA copolymer precursor bearing Gly-Phe-ONp/Gly-Gly-diamine Boclinkers, and addition of LG-PABC linker to PTX and FITC followed bytheir addition to the resulting conjugate HPMAcopolymer-Gly-Phe-Leu-Gly-PABC-FITC-DR1-PTX. The functional monomers andpreparation thereof, for synthesizing HPMA copolymer precursor aredescribed in Example 8 hereinabove and in FIGS. 19A and 19B. Thefunctionalized PTX and FITC moieties and the preparation thereof arepresented in FIGS. 22A-B.

FRET-Based Systems with PGA Conjugates (FRET Mode I):

A PGA-Cy5 conjugate was prepared according to the procedure describedhereinabove (see, Example 2 and FIGS. 8A-B). A Quencher was thereafterconjugated to the polymeric backbone upon activating the unoccupiedcarboxylic groups of PGA with CDI coupling agent in a basic environment,as depicted in FIG. 30.

PGA was synthesized via the N-carboxyanydride (NCA) polymerization ofglutamic acid, as shown in FIGS. 7A-D. The obtained polymer wasdissolved in anhydrous N,N-Dimethylformamide (DMF) and mixed withcarbonyldiimidazole (CDI) coupling reagent in order to activate the freepolymer's carboxyl groups. The reaction mixture was stirred at roomtemperature for 4 hours in basic environment. The activated polymers wasthen added to Cy5-NH₂ (see, FIG. 8A), and the reaction mixture wasstirred overnight at room temperature in basic environment. The reactionwas followed by High Pressure Liquid Chromatography (HPLC) (UltiMate®3000 Nano LC systems, Dionex). At the end of the reaction, theprecipitate was washed with acetone:chloroform (4:1) solution and driedunder vacuum. In order to remove excess of free fluorophore, the driedresidue was dissolved in NaHCO₂ 0.2M buffer and dialyzed for 1 day at 4°C. (MWCO 6-8 kDa) against DI water. The obtained conjugate was purifiedby size exclusion chromatography (SEC) performed using AKTA/FPLC system(Pharmacia/GE Healthcare), HiTrap Desalting columns (Sephadex G-25Superfine) in DDW, flow rate 1.0 ml/min; UV detection. Cy5 loading wasdetermined using SpectraMax M5^(e) multi-detection reader. Theabsorbance of conjugated Cy5 was measured and compared to that of freeCy5.

The obtained PGA-Cy5 conjugate was dissolved in an anhydrous DMF andmixed again with carbonyldiimidazole (CDI) coupling reagent in order toreactivate the unoccupied polymer's carboxyl groups. The reactionmixture was stirred at room temperature for 4 hours. The solution wasremoved to a round bottom flask containing Quencher-NH₂, and thereaction mixture was stirred overnight at room temperature in basicenvironment and was monitored by High Pressure Liquid Chromatography(HPLC) (UltiMate® 3000 Nano LC systems, Dionex). Once the reaction wascompleted, the precipitate was washed with acetone:chloroform (4:1)mixture and dried under vacuum. In order to remove the excess of freeQuencher, the dried residue was dissolved in NaHCO₂ 0.2M buffer anddialyzed for 1 day at 4° C. (MWCO 6-8 kDa) against DI water. The finalpurification of the conjugate by size exclusion chromatography (SEC) wasperformed using AKTA/FPLC system (Pharmacia/GE Healthcare), HiTrapDesalting columns (Sephadex G-25 Superfine) in DDW, flow rate 1.0ml/min; UV detection. Quencher loading was determined using SpectraMaxM5^(e) multi-detection reader.

FRET-based systems with PEG conjugates (FRET mode IV): A FRET-basedprobe-polymer conjugate for use as the diagnostic component in atheranostic system was designed, using Cy5 as a fluorophore, conjugatedto the end (terminus) of polyethylene glycol (PEG) as the polymericnanocarrier. The PEG-Cy5 conjugate was further conjugated via ananalyte-cleavable linker to a quencher, to provide a PEG-Cy5-Qconjugate, forcing a Turn-OFF fluorescent state on the probe. Anexemplary conjugate was designed to undergo specific activation byhydrogen peroxide, which is overproduced by various tumors, andtherefore can be used as analyte for selective activation. Activationturns on a fluorescence signal through separation of the quencher fromPEG-Cy5 conjugate, as depicted in FIG. 31B.

The PEG-Cy5-Q conjugate was synthesized as depicted in FIG. 31A. Inbrief, A FRET-based probe, actuvatable by hydrogen peroxide, wasprepared as described in Redy et al., 2012 (supra), and was dissolved ina minimal amount of DMF. HBTU (4 equivalents) and DIPEA (15 equivalents)were added and the mixture was stirred for 30 minutes. MonofunctionalPEG amine (13 kDa) was dissolved in DMF, heated to 50° C. and then wasadded to the mixture. The reaction was monitored using RP-HPLC. Uponcompletion, the obtained polymeric conjugate was purified usingpreparative HPLC.

The emitted fluorescence signal in the presence and absence of hydrogenperoxide was measured by.

The emitted fluorescence signal in the presence and absence of hydrogenperoxide was determined by incubation with and without hydrogen peroxidein PBS pH 7.4, while monitoring the emission using a spectrofluorometer(λ_(ex)−630 nm, λ_(em)−670 nm). The results are presented in FIGS.32A-B, and show the emission from the probe as a function of time afteraddition of hydrogen peroxide. A significant increase of the emittedfluorescence was observed within minutes after addition of hydrogenperoxide, whereas no change in fluorescence was observed in the absenceof hydrogen peroxide. The conjugate exhibits stable Turn-OFF propertiesin the absence of hydrogen peroxide for several hours. Upon incubationwith hydrogen peroxide, a gradual increase of the fluorescence signalwas observed (i.e. Turn-ON) that reached saturation within approximately3 hours. As shown in FIG. 32B, the signal was 10 times higher than thebackground as measured by HPLC and CRI Maestro™ imaging system.

The in vivo activation was measured following intravenous injection intotumor-bearing mice. Mice bearing U-87 MG tumors were injected with thePEG-Cy5-Q conjugate into the tail vein and the emitted fluorescence wasmonitored immediately following injection. At two minutes postinjection, a strong fluorescence signal was observed in the tumor alone,as shown in FIG. 33, and retained for more than 6 hours (data notshown). These results indicate the selective activation of the conjugateat the tumor site.

Conjugating a therapeutically active agent to a conjugate as describedherein provides a FRET-based PEG theranostic system (FRET mode IV).

Cathepsin B-Cleavable FRET-Based Fluorogenic Moiety (for Use in FRETModes III and IV):

An exemplary FRET-based fluorogenic moiety (probe) which can be attachedto a polymeric backbone by any of the approaches described herein, hasbeen designed and synthesized.

The chemical structure and the activation mechanism of such a FRET-basedprobe is presented in FIG. 34. The probe is composed of Cy5 fluorescentdye attached through a cathepsin B substrate to a quencher dye. Theselected cathepsin B substrate was the dipeptide Phe-Lys. TheNH₂-terminus of the dipeptide was linked to Cy5 and the COOH-terminuswas linked through a short self-immolative spacer to the quencher.Cleavage of the amide linkage after the lysine, followed by1,6-elimination of the self-immolative spacer, results in separationbetween the Cy5 and the quencher. Consequently, a turn-ON NIRfluorescent signal is obtained from the emission of the Cy5 dye.

The chemical synthesis of the FRET-based probe is presented in FIG. 35.A phenylalanine amino acid was initially reacted with1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde)protecting group, and the protected phenylalanine was coupled with4-nitrophenol using N,N′-dicyclohexylcarbodiimide coupling reagent toobtain an ester. The latter was reacted with H-lys(Boc)-OH to give FKdipeptide, which was treated with 4-amino-benzylalcohol in the presenceof N-methyl morpholine and isobutyl-chloroformate to give abenzylalcohol. The alcohol was activated with 4-nitrophenylchloroformate to afford a carbonate. A Quencher moiety was deprotectedwith trifluoroacetic acid and then reacted with the carbonate to afforda carbamate. The carabamate was first deprotected using 2% of hydrazinein solution of DMF and then reacted with a Cy5-NHS derivative. Theobtained crude product was treated with trifluoroacetic acid to removethe Boc-protecting group and to afford the probe.

A detailed synthetic protocol is described in Kisin-Finfer E., et al.,1; 24(11):2453-8; Bioorg Med Chem Lett. 2014.

The turn-ON response of the FRET-based probe upon reaction withcathepsin B was tested. The probe was incubated with cathepsin B inactivity buffer solution (pH=6.0) and the NIR fluorescence emission wasmonitored using a spectrofluorometer. FIG. 36 shows the increase of theemitted fluorescence at wavelength of 670 nm upon incubation of theprobe with cathepsin B, and the non-significant change in fluorescencein the absence of the enzyme.

The capability of the probe to serve as an imaging agent was determinedby assessing the turn-ON response upon reaction with cathepsin B by theCRI Maestro™ imaging system. FIGS. 37A and 37B show the increase of theemitted fluorescence of the probe, 4 hours following the addition ofcathepsin B.

An intravital imaging evaluation of the probes' fluorescence dependencyon endogenous cathepsin B activity was tested by intra-tumoral injectionto cathepsin B-overexpressing 4T1 mammary adenocarcinoma cells. Theprobe was injected intratumorally and the NIR fluorescence emission wasmonitored over 4 hours. The quantification of time-dependentfluorescence signal within the tumor is presented in FIG. 38. As showntherein, the probe presents a distinguishable increase in its NIRfluorescence over 4 hours after the injection into the tumors.

The exemplary FRET probe presented herein can be conjugated by means of,for example, cathepsin B cleavable linker, to a polymeric backbone, withor without a therapeutically active agent. Conjugation can be effectedby attaching the probe to polymeric backbone units (FRET mode III), asexemplified hereinafter, or to the end of a polymeric backbone chain(FRET mode IV).

FRET-Based Systems with HPMA Conjugates (FRET Mode III):

A FRET-based probe such as described hereinabove can be attached to aportion of the backbone units of an HPMA precursor prepared by RAFTpolymerization as described herein, so as to provide a fluorogenicmoiety that generates a fluorescent signal upon cleavage of the FRETprobe.

The synthesis, chemical structure and activation mode of arepresentative example of such a conjugate are presented in FIG. 39.

Alternatively, a FRET-based probe as described herein can be attached toa carboxylic group or any other terminal group of an HPMA precursorprepared by RAFT polymerization as described herein.

Further alternatively, a FRET-based probe as described herein can beattached via a cleavable linker (e.g., cathepsin B-cleavable linker) toa portion of the backbone units of PGA.

Example 10 ICT-Based Systems

Additional fluorogenic probes which can be attached to a polymericbackbone alone or in combination with a therapeutically active agentinclude fluorogenic probes which are activated by Internal ChargeTransfer (ICT). A fluorogenic ICT-based probe is usually composed of adye, masked by a substrate that acts as a trigger, as depicted in FIG.40. The substrate is attached to a functional group of the dye and thus,masks the optical signal by decreasing or interfering with π-electronsconjugation. Removal of the triggering substrate by an analyte or anenzyme of interest results in release of the free dye and activation ofa measurable signal, such as NIR fluorescence.

Fluorogenic ICT-based probes are described, for example, in WO2012/123916. Such probes can be attached, for example, to a portion ofthe polymeric backbone units of HPMA (e.g., by attachment to HPMAprecursor prepared by RAFT polymerization, as described herein).

FIG. 41 is a schematic illustration of the synthesis of HPMAcopolymer-Gly-Gly-Phe-Lys-PABC-PTX-QCy7 by RAFT polymerization ofcopolymer precursor HPMA-Gly-Gly-ONp followed by coupling theretoamine-Phe-Lys-PABC-PTX, and amine-Phe-Lys-PABC-QCy7 as an example forICT-based fluorescent Turn-On moiety.

The exemplary ICT-based probe shown in FIG. 40 has been demonstrated ashighly efficient probe. See, Kisin-Finfer E., et al., 2014 (supra).

Any other fluorogenic ICT-based probes which are described, for example,in WO 2012/123916, can be attached to a portion of the backbone units ofHPMA precursor prepared by RAFT polymerization, as described herein.

Alternatively, such ICT-based probes can be attached to a functional end(terminal) group of the polymeric backbone.

Further alternatively, such ICT-based probes can be attached to aportion of polymeric backbones of PGA, via a cleavable linker, inaccordance with procedures as described herein.

When an ICT-based probe is attached to a polymeric backbone, atherapeutically active agent (drug) can be attached to another portionof the backbone units, so as to provide a theranostic system.

Alternatively, an ICT-based probe which further comprises a drug, andwhich is designed to release both the drug and a fluorescent dye uponcleavage of the trigger unit, can be attached to the polymeric backbone(either to a portion of the backbone units, via a cleavable linker, orto a terminal group of the backbone chain).

An exemplary such modular theranostic drug delivery system, which can beused in combination with a polymeric carrier according to any one of theembodiments described herein, is described herein.

The system is an example of a prodrug based on a latent fluorophore(fluorogenic moiety), which emits a fluorescence signal in the near-IRrange upon activation, and which is further designed to release ananticancer drug such as camptothecin. Such probes can be designed so asto be attached to a polymeric carrier by enzymatically-cleavable linkers(e.g., cathepsin B cleavable linkers as described herein), such thatupon cleavage, the drug is released and a fluorescent signal isgenerated. An exemplary such system, which can be attached, for example,to HPMA copolymer precursor prepared by RAFT polymerization or PGApolymeric backbone as described herein, and its mode of activation, isshown in FIG. 42.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting. In addition, any priority document(s) of this applicationis/are hereby incorporated herein by reference in its/their entirety.

What is claimed is:
 1. A polymeric system comprising a first polymericmoiety comprising a polymeric backbone composed of a plurality ofbackbone units and having attached to at least a portion of saidbackbone units a fluorogenic moiety, said fluorogenic moiety beingattached to said backbone units via a first cleavable linking moietysuch that upon cleavage of said linking moiety, a fluorescent signal isgenerated, the system further comprising a therapeutically active agent,such that: (i) said fluorogenic moiety is attached to one portion ofsaid backbone units and said therapeutically active agent is attached toanother portion of said backbone units; (ii) said therapeutically activeagent forms a part of said fluorogenic moiety; (iii) saidtherapeutically active agent is attached to said first cleavable linkingmoiety; or (iv) the system further comprises a second polymeric moietycomprising a second polymeric backbone composed of a plurality ofbackbone units and having attached to at least a portion of saidbackbone units a therapeutically active agent.
 2. The polymeric systemof claim 1, wherein upon said cleavage, a fluorescent moiety isgenerated.
 3. The polymeric system of claim 2, wherein said fluorescentmoiety is or comprises a cyanine dye.
 4. The polymeric system of claim1, wherein said first cleavable linking moiety is a first biocleavablelinking moiety.
 5. The polymeric system of claim 1, wherein said firstpolymeric moiety further comprises a quenching agent.
 6. The polymericsystem of claim 5, wherein said fluorogenic moiety is attached to oneportion of said backbone units and said quenching agent is attached toanother portion of said backbone units.
 7. The polymeric system of claim1, wherein said first polymeric moiety is represented by Formula IA:

wherein: A₁, A₂ and A₄ are backbone units forming said polymericbackbone; F is said fluorogenic moiety; Q is a quenching agent; L₂ issaid first cleavable lining moiety; S₂ is a first spacer, linking saidfluorogenic moiety to L₂, or is absent; L₄ is a cleavable ornon-cleavable third linking moiety, linking said quenching agent torespective backbone units, or is absent; S₄ is a third spacer linkingsaid quenching agent to said linking moiety L₄, or is absent; w is aninteger having a value such that w/(x+s+w) multiplied by 100 is in therange of from 0 to 99.9; x is an integer having a value such thatx/(x+s+w) multiplied by 100 is in the range of from 0.1 to 100; and s isan integer having a value such that s/(x+s+w) multiplied by 100 is inthe range of from 0 to 99.9, such that each [A₂-L₂-S₂-F] independentlyrepresents a backbone unit having attached thereto the fluorogenicmoiety; each [A₄-L₄-S₄-Q] independently represents a backbone unithaving attached thereto the quenching agent; and each of said backboneunits A₁, A₂ and A₄ is independently a terminal unit, attached to oneother unit, or is attached to two other units, which can be the same ofdifferent.
 8. The polymeric system of claim 7, wherein s is
 0. 9. Thepolymeric system of claim 7, wherein s is a positive integer.
 10. Thepolymeric system of claim 9, wherein a ratio of s to x which is in arange of from 20:1 to 1:20, or from 10:1:10, or from 5:1 to 1:5, or from2:1 to 1:2, or is 1:1.
 11. The polymeric system of claim 5, wherein saidquenching agent forms a part of said fluorogenic moiety.
 12. Thepolymeric system of claim 10, wherein said fluorogenic moiety comprisesa fluorescent moiety linked by said first cleavable linking moiety or bya degradable spacer to said quenching agent.
 13. The polymeric system ofclaim 12, wherein said fluorogenic moiety is represented by formulae IIIor III*:

wherein: the curled line indicates an attachment point to said firstcleavable linking moiety, or to a spacer that is linked to said firstlinking moiety; F* is said fluorescent moiety; Q is said quenchingagent; S′ is a spacer, or is absent; S′″ is a spacer, or is absent; andS″ is a multifunctional spacer which connects said fluorogenic moiety tosaid first cleavable moiety, or to an additional spacer which isconnected to said cleavable linking moiety.
 14. The polymeric system ofclaim 13, wherein at least S″ in Formula III is a degradable spacer. 15.The polymeric system of claim 13, wherein at least S″ and S′ in FormulaIII* is a degradable spacer.
 16. The polymeric system of claim 1,wherein said fluorogenic moiety is attached to one portion of saidbackbone units and said therapeutically active agent is attached toanother portion of said backbone units, and wherein said therapeuticallyactive agent is attached to said backbone units via a second cleavablelinking moiety.
 17. The polymeric system of claim 16, being representedby Formula I:

wherein: A₁, A₂, A₃ and A₄ are each backbone units covalently linked toone another and forming said polymeric backbone; D is saidtherapeutically active agent; F is said fluorogenic moiety; Q is saidquenching agent; L₂ is said first linking moiety; L₃ is said secondlinking moiety or absent; L₄ is a linking moiety linking said quenchingagent, or is absent; each of S₂, S₃ and S₄ is independently a spacer orabsent; w is an integer having a value such that w/(x+y+w+s) multipliedby 100 is in the range of from 0 to 99.9; x is an integer having a valuesuch that x/(x+y+w+s) multiplied by 100 is in the range of from 0.1 to100; y is an integer having a value such that y/(x+y+w+s) multiplied by100 is in the range of from 0 to 99.9; and s is an integer having avalue such that s/(x+y+w+s) multiplied by 100 is in the range of from 0to 99.9, such that each [A₃-L₃-S₃-D] independently represents a backboneunit having attached thereto said therapeutically active agent; each[A₂-L₂-S₂-F] independently represents a backbone unit having attachedthereto said fluorogenic moiety; and each [A₄-L₄-S₄-Q] independentlyrepresents a backbone unit having attached thereto said quenching agent,wherein when s is 0, said quenching agent forms a part of saidfluorogenic moiety, and when y is 0, said therapeutically active agentforms a part of said fluorogenic moiety.
 18. The polymeric system ofclaim 16, wherein said second linking moiety is a biocleavable linkingmoiety.
 19. The polymeric system of claim 1, wherein the system furthercomprises a second polymeric moiety comprising a second polymericbackbone composed of a plurality of backbone units and having attachedto at least a portion of said backbone units a therapeutically activeagent, and wherein said therapeutically active agent is attached to saidbackbone units via a second cleavable linking moiety.
 20. The polymericsystem of claim 19, wherein said second linking moiety is a biocleavablelinking moiety.
 21. The polymeric system of claim 1, wherein saidtherapeutically active agent forms a part of said fluorogenic moiety, oris attached to said first cleavable linking moiety, and wherein uponsaid cleavage, said therapeutically active agent is released.
 22. Thepolymeric system of claim 21, wherein upon said cleavage, a fluorescentmoiety is generated.
 23. The polymeric system of claim 22, wherein saidfluorescent moiety is or comprises a cyanine dye.